ANTI-RESPIRATORY SYNCYTIAL VIRUS (RSV) ANTIBODY CELL-BASED POTENCY ASSAY

Abstract

The disclosure describes methods for measuring the potency of anti-Respiratory Syncytial Virus (RSV) antibody against respiratory syncytial virus in a cell culture system using an RSV reporter virus. The disclosure also describes respiratory syncytial viruses that carry a reporter gene, and expression vectors for producing an infectious recombinant respiratory syncytial virus (RSV) that carry a reporter gene.

Description

FIELD OF THE INVENTION

The present disclosure describes a cell-based potency assay for measuring anti-Respiratory Syncytial Virus (RSV) antibody activity using a cultured system with A549 cells and an RSV reporter virus.

BACKGROUND OF THE INVENTION

The human RSV genome is a single-stranded negative-sense RNA molecule of approximately 15 kb that encodes 11 proteins. Two of these proteins are the main surface glycoproteins of the virion. These are (i) the attachment (G) protein, which mediates virus binding to cells, and (ii) the fusion (F) protein, which promotes both fusion of the viral and cell membranes at the initial stages of the infectious cycle and fusion of the membrane of infected cells with those of adjacent cells to form characteristic syncytia. The attachment protein G binds cellular surface receptors and interacts with F. This interaction triggers a conformational change in F to induce membrane fusion, thereby releasing the viral ribonucleoprotein complex into the host cell cytoplasm.

Monoclonal antibodies against the F protein or the G protein have been shown to have neutralizing effect in vitro and prophylactic effects in vivo. See, e.g., Beeler and Coelingh 1989, J. Virol. 63:2941-50; Garcia-Barreno et al., 1989, J. Virol. 63:925-32; Taylor et al., 1984, Immunology 52: 137-142; Walsh et al., 1984, Infection and Immunity 43:756-758; and U.S. Pat. Nos. 5,842,307 and 6,818,216. Neutralizing epitopes on the F glycoprotein were originally mapped by identifying amino acids that were altered in antibody escape variants and by assessing antibody binding to RSV F-derived peptides.

Several anti-RSV antibodies are in clinical development for use as a passive immunotherapy agent to protect against RSV infection in infants and the elderly who have immature or compromised immune systems. Such anti-RSV antibodies must be highly potent in order to act effectively as passive immunotherapy agents. Thus, there is a need for RSV neutralization assay as a cell-based potency assay, for example, to confirm potency of clinical batches of anti-RSV antibodies.

The industry standard of virus neutralization assay is the plaque reduction neutralization test (PRNT) (McKimm-Breschkin J L. 2004) . The PRNT is based on manual plaque counting and is low throughput, requires significant resources, and has high variability. Thus, an assay for determining the potency of anti-RSV antibodies that is fast, easy to use, and has low variability would be an asset for the development of anti-RSV antibody clinical candidates.

SUMMARY OF THE INVENTION

The present invention provides respiratory syncytial viruses engineered to encode reporter genes, nucleic acids encoding such viruses, and cell-based potency assays for measuring anti-RSV antibody activity using a cell culture system.

In one aspect, the disclosure provides an expression vector for producing an infectious recombinant respiratory syncytial virus (RSV) comprising: a) a nucleic acid sequence encoding a respiratory syncytial virus; and b) a reporter gene flanked by an RSV gene start sequence and an RSV gene end sequence, the reporter gene and flanking RSV gene start and RSV gene end sequences located between the P and M genes of the respiratory syncytial virus.

In another aspect, the disclosure provides a respiratory syncytial virus comprising: a) a nucleic acid sequence encoding a respiratory syncytial virus; and b) a reporter gene flanked by an RSV gene start sequence and an RSV gene end sequence, the reporter gene and flanking RSV gene start and RSV gene end sequences located between the P and M genes of the respiratory syncytial virus.

In some embodiments of the foregoing expression vector and respiratory syncytial virus, a) the reporter gene encodes a luminescent enzyme that catalyzes a luminescent substrate; or b) the reporter gene encodes a fluorescent protein. In some embodiments, the luminescent enzyme is a luciferase. In some embodiments, the RSV gene start sequence is SEQ ID NO: 16 and the RSV gene end sequence is SEQ ID NO: 17. In some embodiments, the sequence of the reporter gene is SEQ ID NO: 18. In some embodiments, the reporter gene flanked by an RSV gene start sequence and an RSV gene end sequence is SEQ ID NO: 15. In some embodiments, the respiratory syncytial virus is strain A2 or comprises a nucleic acid of SEQ ID NO: 14.

In another aspect, the disclosure provides a method for measuring the activity of an anti-respiratory syncytial virus (RSV) antibody or antigen binding fragment thereof, the method comprising the steps of: a) combining (i) the anti-RSV antibody or antigen binding fragment thereof, (ii) an RSV virus comprising a reporter gene, and (iii) one or more cells infectable by the RSV virus; and b) detecting expression of the reporter gene.

In some embodiments of the foregoing method, the one or more cells infectable by the RSV virus are combined with the anti-RSV antibody or antigen binding fragment thereof before the RSV virus is added. In some embodiments, a) the reporter gene encodes a luminescent enzyme that catalyzes a luminescent substrate, and detecting expression of the reporter gene comprises detecting luminescence of the luminescent substrate; or b) the reporter gene encodes a fluorescent protein, and detecting expression of the reporter gene comprises detecting fluorescent light emission from the fluorescent protein. In some embodiments, the reporter gene is a luminescent enzyme. In some embodiments, the luminescent enzyme is a luciferase. In some embodiments, the reporter gene is a fluorescent protein. In some embodiments, the fluorescent protein is a protein excited in the UV wavelength, such as Sirius, Sandercyanin, shBFP-N158S/L173I. In some embodiments, the fluorescent protein is a protein excited by a blue wavelength of light, such as Azurite, EBFP2, mKalama1, mTagBFP2, TagBFP, or shBFP. In some embodiments, the fluorescent protein is a protein excited by a cyan wavelength of light, such as ECFP, Cerulean, mCerulean3, SCFP3A, CyPet, mTurquoise, mTurquoise2, TagCFP, mTFP1, monmeric Midoriishi-Cyan, or Aquamarine. In some embodiments, the fluorescent protein is a protein excited by a green wavelength of light, such as GFP, TurboGFP, TagGFP2, mUKG, Superfolder GFP, Emerald, EGFP, Monomeric Azami Green, mWasabi, Clover, mNeonGreen, NowGFP, or mClover3. In some embodiments, the fluorescent protein is a protein excited by a yellow wavelength of light, such as TagYFP, EYFP, Topaz, Venus SYFP2, Citrine, Ypet, IanRFP-deltaS83, mPapayal, or mCyRFP1. In some embodiments, the fluorescent protein is a protein excited by an orange wavelength of light, such as Monomeric Kusabira-Orange, mOrange, mOrange2, mKOkappa, or mKO2, In some embodiments, the fluorescent protein is a protein excited by a red wavelength of light, such as TagRFP, TagRFP-T, RRvT, mRuby, mRuby2, mTangerine, mApple, mStrawberry, FusionRed, mCherry, mNectarine, mRuby3, mScarlet, or mScarlet-I. In some embodiments, the fluorescent protein is a protein excited by a far-red wavelength of light, such as mKate2, hcRed-Tandem, mPlum, mRaspberry, mNeptune, NirFP, TagRFP657, TagRFP675, mCardinal, mStable, mMaroon1, or mGarnet2. In some embodiments, the fluorescent protein is a protein excited by a near infra-red wavelength of light, such as iFP1.4, iRFP713 (iRFP), iRFP670, iRFP682, iRFP702, iRFP720, iFP2.0, mIFP, TDsmURFP, or miRFP670. In some embodiments, the fluorescent protein is Sapphire, T-Sapphire, or mAmetrine. In some embodiments, the fluorescent protein has a long Stokes shift, such as mKeima, mBeRFP, LSS-mKate2, LSS-mKate1, LSSmOrange, CyOFP1, or Sandercyanin.

In some embodiments of the foregoing method, the method further comprises a step of adding a luciferase substrate.

In some embodiments of the foregoing method, the one or more cells infectable by the RSV virus are A549 cells.

In another aspect, the disclosure provides a method for measuring the activity of an anti-respiratory syncytial virus (RSV) antibody or antigen binding fragment thereof, the method comprising the steps of: a) combining the anti-RSV antibody or antigen-binding fragment thereof with an RSV virus comprising a nucleic acid sequence encoding a luciferase; b) adding the mixture of step a) to one or more A549 cells; c) adding a luciferase substrate to the mixture of step b); and d) detecting luminescence of the luciferase substrate.

In some embodiments of any one of the foregoing methods wherein the reporter gene is a luciferase, the nucleic acid sequence encoding the luciferase encodes a nanoluciferase.

In some embodiments of any one of the foregoing aspects, the anti-RSV antibody or antigen binding fragment thereof comprises: (a) three heavy chain complementarity determining regions (HC-CDRs), wherein HC-CDR1 is SEQ ID NO: 1, HC-CDR2 is SEQ ID NO: 2, and HC-CDR3 is SEQ ID NO: 3; and (b) three light chain complementarity determining regions (LC-CDRs), wherein LC-CDR1 is SEQ ID NO: 4, LC-CDR2 is SEQ ID NO: 5, and LC-CDR3 is SEQ ID NO: 6. In some embodiments, the anti-RSV antibody or antigen binding fragment thereof comprises a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8. In some embodiments, the anti-RSV antibody or antigen binding fragment thereof comprises a heavy chain and a light chain, and wherein the heavy chain comprises SEQ ID NO: 9 and the light chain comprises SEQ ID NO: 10. In some embodiments, the anti-RSV antibody or antigen binding fragment thereof is an antibody comprising two heavy chains of SEQ ID NO: 9 and two light chains of SEQ ID NO: 10.

In some embodiments of the foregoing methods, the RSV virus comprises a) a nucleic acid sequence encoding a respiratory syncytial virus; and b) a reporter gene flanked by an RSV gene start sequence and an RSV gene end sequence, the reporter gene and flanking RSV gene start and RSV gene end sequences located between the P and M genes of the respiratory syncytial virus. In some embodiments, a) the reporter gene encodes a luminescent enzyme that catalyzes a luminescent substrate, and detecting expression of the reporter gene comprises detecting luminescence of the luminescent substrate; or b) the reporter gene encodes a fluorescent protein, and detecting expression of the reporter gene comprises detecting fluorescent light emission from the fluorescent protein. In some embodiments, the luminescent enzyme is a luciferase. In some embodiments, the RSV gene start sequence is SEQ ID NO: 16 and the RSV gene end sequence is SEQ ID NO: 17. In some embodiments, the sequence of the reporter gene is SEQ ID NO: 18. In some embodiments, the reporter gene flanked by an RSV gene start sequence and an RSV gene end sequence is SEQ ID NO: 15. In some embodiments, the respiratory syncytial virus is strain A2 or comprises a nucleic acid of SEQ ID NO: 14.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of a cell-based potency assay with anti-RSV Antibody A.

FIGS. 2A-2C show exemplary dilution plate maps. Maps for plates 1-3 are shown in FIGS. 2A, 2B and 2C, respectively.

FIG. 3 shows a graph of a typical result of cell-based potency assay for Antibody A.

DETAILED DESCRIPTION

Disclosed herein is a cell-based potency assay for measuring anti-RSV antibody activity using a cultured system with cells including for example A549 cells. In one embodiment, the assay utilizes an RSV reporter virus, RSV-NLucP, which encodes a gene for NanoLuc® luciferase enzyme within the RSV genome. After infecting cells, the RSV-NLucP expresses the NanoLuc® luciferase, which generates luminescence signal upon the addition of the luciferase substrate. Luminescence signal is directly proportional to RSV-NLucP infectivity. Introduction of anti-RSV antibody blocks the entry of RSV-NLucP into the host cells and therefore inhibit luminescence signal. In one embodiment, the anti-RSV antibody is Antibody A having two heavy chains of SEQ ID NO: 9 and two light chains of SEQ ID NO: 10. The assay is robust, quantitative and accurate.

Certain technical and scientific terms are defined below. Unless specifically defined elsewhere in this specification, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise.

As used herein, the term “antibody” refers to any form of antibody that exhibits the desired biological or binding activity. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized, fully human antibodies, chimeric antibodies and camelized single domain antibodies. “Parental antibodies” are antibodies obtained by exposure of an immune system to an antigen prior to modification of the antibodies for an intended use, such as humanization of an antibody for use as a human therapeutic.

In general, the basic (or “full-length”) antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989). In the context of an antibody or antigen binding fragment thereof, the terms “domain” and “region” can be used interchangeably, where appropriate.

The variable regions of each light/heavy chain pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are, in general, the same.

Typically, the variable domains of both the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), which are located within relatively conserved framework regions (FR). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C-terminal, both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5^th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

As used herein, unless otherwise indicated, “antibody fragment” or “antigen binding fragment” refers to antigen binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions. Examples of antibody binding fragments include, but are not limited to, Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.

An antibody that “specifically binds to” a specified target protein is an antibody that exhibits preferential binding to that target as compared to other proteins, but this specificity does not require absolute binding specificity. An antibody is considered “specific” for its intended target if its binding is determinative of the presence of the target protein in a sample, e.g. without producing undesired results such as false positives. Antibodies, or binding fragments thereof, useful in the present invention will bind to the target protein with an affinity that is at least two fold greater, preferably at least ten times greater, more preferably at least 20-times greater, and most preferably at least 100-times greater than the affinity with non-target proteins. As used herein, an antibody is said to bind specifically to a polypeptide comprising a given amino acid sequence, e.g. the amino acid sequence of an RSV antigen, if it binds to polypeptides comprising that sequence but does not bind to proteins lacking that sequence.

“Chimeric antibody” refers to an antibody in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in an antibody derived from a particular species (e.g., human) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in an antibody derived from another species (e.g., mouse) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

“Human antibody” refers to an antibody that comprises human immunoglobulin protein sequences only. A human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” or “rat antibody” refer to an antibody that comprises only mouse or rat immunoglobulin sequences, respectively.

“Humanized antibody” refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The prefix “hum”, “hu” or “h” is added to antibody clone designations when necessary to distinguish humanized antibodies from parental rodent antibodies. The humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody, or for other reasons.

“Comprising” or variations such as “comprise”, “comprises” or “comprised of” are used throughout the specification and claims in an inclusive sense, i.e., to specify the presence of the stated features but not to preclude the presence or addition of further features that may materially enhance the operation or utility of any of the embodiments of the invention, unless the context requires otherwise due to express language or necessary implication.

“Conservatively modified variants” or “conservative substitution” refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that the changes can frequently be made without altering the biological activity or other desired property of the protein, such as antigen affinity and/or specificity. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benj amin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are set forth in Table 1 below.

TABLE 1
Exemplary Conservative Amino Acid Substitutions
Original residue Conservative substitution
Ala (A)          Gly; Ser
Arg (R)          Lys; His
Asn (N)          Gln; His
Asp (D)          Glu; Asn
Cys (C)          Ser; Ala
Gln (Q)          Asn
Glu (E)          Asp; Gln
Gly (G)          Ala
His (H)          Asn; Gln
Ile (I)          Leu; Val
Leu (L)          Ile; Val
Lys (K)          Arg; His
Met (M)          Leu; Ile; Tyr
Phe (F)          Tyr; Met; Leu
Pro (P)          Ala
Ser (S)          Thr
Thr (T)          Ser
Trp (W)          Tyr; Phe
Tyr (Y)          Trp; Phe
Val (V)          Ile; Leu

“Consists essentially of,” and variations such as “consist essentially of” or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited elements or group of elements, and the optional inclusion of other elements, of similar or different nature than the recited elements, that do not materially change the basic or novel properties of the specified dosage regimen, method, or composition. As a non-limiting example, an anti-RSV antibody that consists essentially of a recited amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues, which do not materially affect the properties of the binding compound.

“Framework region” or “FR” as used herein means the immunoglobulin variable regions excluding the CDR regions.

“Kabat” as used herein means an immunoglobulin alignment and numbering system pioneered by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.).

“Anti-RSV antibody” means an antibody or antigen-binding fragment thereof that binds human RSV F protein, preferably from both RSV A strains and B strains, that binds both the pre-fusion F protein and the post-fusion F protein. In some embodiments, the anti-RSV F-protein antibodies are isolated. The antibodies described herein bind to an epitope at site IV of the F protein. In any of the embodiments of the invention described herein, in certain embodiments, the heavy chain or heavy chain variable region does not comprise the amino acid sequence of SEQ ID NO: 9 and/or the light chain or light chain variable region does not comprise the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the heavy chain comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 23 and the light chain comprises, consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 25.

In one embodiment, the anti-RSV F-protein antibodies are fully human.

As used herein, an anti-RSV F-protein antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof that specifically binds to human RSV F protein. An antibody or antigen-binding fragment thereof that “specifically binds to human RSV” is an antibody or antigen-binding fragment thereof that binds to the pre-fusion or post-fusion human RSV F protein with a Kd of about 1 nM or a higher affinity (e.g., 1 nM-2 pM, 1 nM, 100 pM, 10 pM or 2 pM), but does not bind to other proteins lacking RSV F protein sequences. In one embodiment, the antibody of the invention which specifically binds to human RSV F protein is also cross-reactive with bovine RSV F protein. As used herein “cross-reactivity” refers to the ability of an antibody to react with a homologous protein from other species. Whether an antibody specifically binds to human RSV F protein can be determined using any assay known in the art. Examples of assays known in the art to determining binding affinity include surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g., KinExa or OCTET).

In some embodiments of any of the foregoing aspects, the anti-RSV antibody or antigen binding fragment thereof is a “Fab fragment”. A “Fab fragment” is comprised of one light chain and the C_H1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A “Fab fragment” can be the product of papain cleavage of an antibody.

In some embodiments of any of the foregoing aspects, the anti-RSV antibody or antigen binding fragment thereof comprises an Fc region. An “Fc” region contains two heavy chain fragments comprising the C_H1 and C_H2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the C_H3 domains.

In some embodiments of any of the foregoing aspects, the anti-RSV antibody or antigen binding fragment thereof is a Fab′ fragment. A “Fab′ fragment” contains one light chain and a portion or fragment of one heavy chain that contains the V_H domain and the C_H1 domain and also the region between the C_H1 and C_H2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab′ fragments to form a F(ab′)₂ molecule.

In some embodiments of any of the foregoing aspects, the anti-RSV antibody or antigen binding fragment thereof is a F(ab′)₂ fragment. A “F(ab′)₂ fragment” contains two light chains and two heavy chains containing a portion of the constant region between the C_H1 and C_H2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′ fragments that are held together by a disulfide bond between the two heavy chains. A “F(ab′)₂ fragment” can be the product of pepsin cleavage of an antibody.

In some embodiments of any of the foregoing aspects, the anti-RSV antibody or antigen binding fragment thereof is a Fv fragment. The “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions. In some embodiments of any of the foregoing aspects, the anti-RSV antibody or antigen binding fragment thereof is a scFv fragment. The term “single-chain Fv” or “scFv” antibody refers to antibody fragments comprising the V_H and V_L domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the V_H and V_L domains which enables the scFv to form the desired structure for antigen-binding. For a review of scFv, see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also, International Patent Application Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203.

In some embodiments of any of the foregoing aspects, the anti-RSV antibody or antigen binding fragment thereof is a domain antibody. A “domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more V_H regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two V_H regions of a bivalent domain antibody may target the same or different antigens.

In some embodiments of any of the foregoing aspects, the anti-RSV antibody or antigen binding fragment thereof is a bivalent antibody. A “bivalent antibody” comprises two antigen-binding sites. In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific (see below).

In some embodiments of any of the foregoing aspects, the anti-RSV antibody or antigen binding fragment thereof is a diabody. As used herein, the term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V_H) connected to a light chain variable domain (V_L) in the same polypeptide chain (V_H-V_L or V_L-V_H). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a review of engineered antibody variants generally see Holliger and Hudson (2005) Nat. Biotechnol. 23 :1126-1136.

Typically, an antibody or antigen-binding fragment as described herein which is modified in some way retains at least 10% of its binding activity (when compared to the parental antibody) when that activity is expressed on a molar basis. Preferably, an antibody or antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the RSV F-protein binding affinity as the parental antibody. It is also intended that an antibody or antigen-binding fragment can include conservative or non-conservative amino acid substitutions (referred to as “conservative variants” or “function conserved variants” of the antibody) that do not substantially alter its biologic activity.

In some embodiments of any of the foregoing aspects, the anti-RSV antibodys or antigen binding fragments thereof are isolated anti-hRSV F-protein antibodies and antigen-binding fragments thereof. “Isolated” antibodies or antigen-binding fragments thereof are at least partially free of other biological molecules from the cells or cell cultures in which they are produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term “isolated” is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments.

In some embodiments of any of the foregoing aspects, the anti-RSV antibodys or antigen binding fragments thereof are monoclonal anti-hRSV F-protein antibodies and antigen-binding fragments thereof. The term “monoclonal antibody” or “mAb” or “Mab”, as used herein, refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains, particularly their CDRs that are often specific for different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597, for example. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

In one embodiment, the anti-RSV antibody or antigen-binding fragment thereof is Antibody A, comprising two heavy chains of SEQ ID NO: 9 and two light chains of SEQ ID NO: 10.

In one embodiment, the anti-RSV antibody or antigen-binding fragment thereof comprises a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10.

In one embodiment, the anti-RSV antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 7 and the light chain variable region comprising SEQ ID NO: 8.

As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody or antigen-binding fragment thereof that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a “complementarity determining region” or “CDR” (i.e. CDRL1, CDRL2 and CDRL3 in the light chain variable domain and CDRH1, CDRH2 and CDRH3 in the heavy chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining the CDR regions of an antibody by sequence); see also Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917 (defining the CDR regions of an antibody by structure). As used herein, the term “framework” or “FR” residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

“Isolated nucleic acid molecule” or “isolated polynucleotide” means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. For purposes of this disclosure, it should be understood that “a nucleic acid molecule comprising” a particular nucleotide sequence does not encompass intact chromosomes. Isolated nucleic acid molecules “comprising” specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and/or may include vector sequences.

A “virus”, “viral particle”, “virus particle”, or “recombinant infectious virus particle” as used herein refers to a single particle derived from a viral nucleic acid, which is located outside a cell. The viral particle thus represents the mature and infectious form of a virus. As the viral particle contains genetic information, it is able to replicate and/or it can be propagated in a susceptible host cell. Depending on the complexity of a virus, the viral particle comprises nucleic acid and polypeptide sequences and, optionally lipids, preferably in the form of a lipid membrane derived from the host cell. A virus having nucleic acid sequences may include genes encoding non-native proteins, called a “genetic payload”

A “polynucleotide sequence”, “nucleic acid sequence” or “nucleotide sequence” is a series of nucleotide bases (also called “nucleotides”) in a nucleic acid, such as DNA or RNA, and means any chain of two or more nucleotides.

The nucleic acids herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5′- and 3′-non-coding regions, and the like.

A “coding sequence” or a sequence “encoding” an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in production of the product.

The term “gene” means a DNA sequence that codes for or corresponds to a particular sequence of ribonucleotides or amino acids which comprise all or part of one or more RNA molecules, proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine, for example, the conditions under which the gene is expressed. Genes may be transcribed from DNA to RNA which may or may not be translated into an amino acid sequence.

In general, a “promoter” or “promoter sequence” is a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. A promoter sequence is, in general, bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at any level. Within the promoter sequence may be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The promoter may be operably associated with other expression control sequences, including enhancer and repressor sequences or with a reporter gene.

A coding sequence is “under the control of”, “functionally associated with”, “operably linked”, or “operably associated with” transcriptional and translational control sequences in a cell when the sequences direct RNA polymerase mediated transcription of the coding sequence into RNA, preferably mRNA, which then may be RNA spliced (if it contains introns) and, optionally, translated into a protein encoded by the coding sequence.

The terms “express” and “expression” mean allowing or causing the information in a gene, RNA or DNA sequence to become manifest; for example, producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene. A DNA sequence is expressed in or by a cell to form an “expression product” such as an RNA (e.g., mRNA) or a protein. The expression product itself may also be said to be “expressed” by the cell.

The term “upstream” refers that the gene is to the 5′ end of the other gene.

The term “transformation” means the introduction of a nucleic acid into a cell. The introduced gene or sequence may be called a “clone”. A host cell that receives the introduced DNA or RNA has been “transformed” and is a “transformant” or a “clone.” The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or from cells of a different genus or species.

A “reporter gene” is a gene encoding a protein that is detectable by fluorescence, luminescence, color change, enzyme assay, or histochemistry. For example, a fluorescent reporter protein encoded by a reporter gene may be a fluorescent protein that fluoresces when exposed to a certain wavelength of light (e.g., GFP). A reporter protein may be a reporter enzyme that catalyzes a reaction with a substrate to produce an observable change in that substrate. Enzymes such as luciferase (exemplary substrate luciferin) or β-lactamase (exemplary substrate CCF4) can cause luminescence or allow fluorescence on substrate cleavage, and enzymes such as β-galactosidase (exemplary substrate X-gal (5-bromo-4-chloro- 3-indolyl-P-D-galactopyranoside)) and secreted alkaline phosphatase (exemplary substrate PNPP (p-Nitrophenyl Phosphate, Disodium Salt)) can result in a visualizable precipitate upon substrate cleavage. The term “luminescent substrate” is a substrate that luminesces upon catalysis by a reporter enzyme, e.g. luciferin. The term “luminescent enzyme” or “luminescent reporter enzyme” refers to an enzyme that catalyzes a reaction with a luminescent substrate. In some embodiments, a reporter protein is detectable by an antibody binding interaction.

The term “luminescent substrate” refers to a substrate that luminesces upon catalysis by a reporter enzyme, e.g. luciferin. The term “luminescent enzyme” or “luminescent reporter enzyme” refers to an enzyme that catalyzes a reaction with a luminescent substrate, e.g. luciferase or nanoluciferase. Various luciferase genes encoding luciferase are commercially available for use in the invention, including luciferase genes from fireflies, sea pansy (Renilla reniformis), ostracods (Cypridina hilgendorfii), and copepods (Gaussia princeps) (see Bioconjug Chem. 2016 May 18; 27(5): 1175-1187, incorporated herein by reference). One luciferase enzyme is NanoLuc® (also called Nluc), a modified 19 kDa luciferase derived from deep sea shrimp (Oplophorus gracihrostris) that can be purchased from Promega Corp. Nluc uses the substrate furimazine to produce high intensity, glow-type luminescence when expressed in cells.

The term “fluorescent protein” refers to a protein that emits light at some wavelength after excitation by light at another wavelength. Exemplary fluorescent proteins that emit in the green spectrum range include, but are not limited to: green fluorescent protein (GFP); enhanced GFP (eGFP); superfolder GFP; AcGFP1; and ZsGreen1. Exemplary fluorescent proteins that emit light in the blue spectrum range include, but are not limited to: enhanced blue fluorescent protein (EBFP), EBFP2, Azurite, and mKalama. Exemplary fluorescent proteins that emit light in the cyan spectrum range include, but are not limited to: cyan fluorescent protein (CFP); enhanced CFP (ECFP); Cerulean; mHoneydew; and CyPet. Exemplary fluorescent proteins that emit light in the yellow spectrum range include, but are not limited to: yellow fluorescent protein (YFP); Citrine; Venus; mBanana; ZsYellow 1; and Ypet. Exemplary fluorescent proteins that emit in the orange spectrum range include, but are not limited to: mOrange; tdTomato; Exemplary fluorescent proteins that emit light in the red and far-red spectrum range include, but are not limited to: DsRed; DsRed-monomer; DsRed-Express2; mRFPi; mCherry; mStrawberry; mRaspberry; niPluni; E2-Crimson; iRFP670; iRFP682; iRFP702; iRFP720. Exemplary listings of fluorescent proteins and their characteristics may be found in Day and Davidson, Chem Soc Rev 2009 October; 38(10): 2887-2921, incorporated herein by reference.

Fluorescent proteins may include chimeric combinations of fluorescent proteins that transfer and receive energy through fluorescent resonance energy transfer (FRET) when exposed to a particular wavelength of light. In some embodiments, an acceptor in a FRET pair may emit light at a certain wavelength after accepting energy from a donor molecule exposed to another wavelength of light. Exemplary chimeric FRET pairs, include, but are not limited to ECFP-EYFP; mTurquoise2-SeYFP; EGFP-mCherry; and Clover-mRuby. In some embodiments, the acceptor molecule of chimeric fluorescent molecule may quench the light emission of a donor molecule exposed to its preferred wavelength of light. Quenching between different portions of chimeric fluorescent proteins may occur using a photoactivatable acceptor. For example, a chimeric fluorescent protein may include a photoactivatable GFP that can then quench photoemission by CFP. Examples of FRET proteins are discussed in Ehldebrandt et al., Sensors (Basel). 2016 September; 16(9): 1488, incorporated herein by reference.

“Read-out signal” refers to a signal produced from the reporter gene protein expression. The signal can be emitted by the protein or reaction of the protein with a substrate. In one embodiment, the signal is fluorescence or luminescence.

“Stably transfected” refers to the foreign gene being part of the host genome and is therefore replicated. This is typically initiated by transiently transfecting a cell with the foreign gene but through a process of careful selection and amplification, and stable clones are generated. One method to select for stable clones is to use selectable markers expressed on the plasmid DNA to enable the selection of any cells that have successfully integrated the gene into their genome. A common method used is to design the plasmid DNA to also contain a gene that expresses antibiotic resistance. Continued antibiotic treatment of the cells for long-term results in the expansion of only the stably-transfected cells. Descendants of these stably-transfected cells, also express the foreign gene, resulting in a stably transfected cell line.

The term “vector” includes a vehicle (e.g., a plasmid) by which a DNA or RNA sequence can be introduced into a host cell, so as to transform the host and, optionally, promote expression and/or replication of the introduced sequence.

“Assay media” refers to a solution comprising a nutrient(s) for cells such as glucose, vitamins, amino acids, or a combination thereof and serum, and optionally antibiotics and a buffer.

In some embodiments, the A549 stable cell line is used to test anti-RSV antibodies in an anti-RSV functional cell-based assay. The A549 cell line is a lung epithelial cell line derived from a human carcinoma.

GENERAL METHODS

Standard methods in molecular biology are described in Sambrook, Fritsch and Maniatis (1982 & 1989 2^nd Edition, 2001 3^rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protcols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand/receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

Monoclonal, polyclonal, and humanized antibodies can be prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

An alternative to humanization is to use human antibody libraries displayed on phage or human antibody libraries in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995) Nature Medicine 1:837 -839; Mendez et al. (1997) Nature Genetics 15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, CA; de Bruin et al. (1999) Nature Biotechnol. 17:397-399).

Purification of antigen is not necessary for the generation of antibodies. Animals can be immunized with cells bearing the antigen of interest. Splenocytes can then be isolated from the immunized animals, and the splenocytes can fuse with a myeloma cell line to produce a hybridoma (see, e.g., Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242; Preston et al., supra; Kaithamana et al. (1999) J. Immunol. 163:5157-5164).

Methods for flow cytometry, including fluorescence activated cell sorting (FACS), are available (see, e.g., Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2^nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are available (Molecular Probesy (2003) Catalogue, Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis, MO).

EXAMPLES

Definitions for the abbreviations used herein are provided below.

• • BSC Biological Safety Cabinet
  • BSL Bio Safety Level
  • CDR Complementarity determining region
  • COA Certificate of Analysis
  • Conc. Concentration
  • DP Drug Product
  • DS Drug Substance
  • ELN Electronic Laboratory Notebook
  • FBS HI Fetal Bovine Serum, Heat Inactivated
  • h hour(s)
  • HC heavy chain
  • LC light chain
  • mg Milligram
  • min/min. Minute(s)
  • mL Milliliter
  • No. Number
  • RPM Revolutions Per Minute
  • RSV Respiratory Syncytial Virus
  • RSV-NLucP Respiratory Syncytial Virus-Nanoluc Luciferase-PEST
  • RT Room Temperature
  • SDS Safety Data Sheets
  • SOP Standard Operating Procedure
  • TC-treated Tissue Culture-Treated
  • Temp. Temperature
  • uL/μL Micro liter
  • WCB Working Cell Bank
  • WVS Working Virus Seed

Equipment used in the examples described here are listed in Table 1 below. Equivalent equipment can also be used.

TABLE 1
Name	Manufacturer	Model/Part No.
a. Biological Safety Cabinet, Class II Type A2	The Baker	SterilGARD
	Company	III
b. Humidified CO2 Incubator, 37 ± 1° C., 5 ± 1% CO2	Thermo Fisher	Steri-Cult
	Scientific
c. Centrifuge with Swing Rotor (for 15- and 50-mL	Beckman	Allegra 6R
tubes)	Coulter
d. Light Inverted Microscope	Zeiss	Invertoskop
e. Vi-Cell Automated Cell Counter	Beckman	Vi-Cell XR
	Coulter
f. C-Chip Disposable Hemocytometer (for Manual	SKC, Inc.	22-600-107
Cell Counting, optional)
g. Refrigerator (5 ± 3° C.)	Any Suitable	NA
h. Freezer (−20 ± 5° C.)	Any Suitable	NA
i. Ultra-Low Freezer (−80 ± 10° C.)	Any Suitable	NA
j. Water Bath (37 ± 2° C.)	Thermo Fisher	Isotemp 210
	Scientific
k. Vortexer	Any Suitable	NA
l. Microtiter Plate Shaker	Thermo Fisher	4625
	Scientific
m. Wellpro 3000-PG3025 (for serial dilution and	Wellpro Group	PG3025
reagent transfer, optional)
n. Viaflo 384 Handheld Electronic 24, 96 and 384	Integra	18030703
Channel Pipette (for reagent addition or transfer, optional)
o. SpectraMax Plate Reader	Molecular	M5 or M5E
	Devices

Consumables and Materials used in the examples are provided in Table 2 below.

TABLE 2
Name	Manufacturer	Model/Part No.
a. PET Media Bottle, sterile, 250 mL	Thermo Fisher	342040-0250
	Scientific
b. 0.2 μm Nalgene Rapid Flow Sterile Filter	Thermo Fisher	566-0020 and 567-
Unit with PES Membrane, 500 mL and 1000 mL	Scientific	0020
c. 96-well Polypropylene Round Bottom	Corning	3879
Microplates with Polystyrene Lids, Sterile
d. 96-well white polystyrene CellBind	Corning or	3809 (CellBIND) or
Microplate, sterile, flat bottom clear wells	PerkinElmer	6005181
		(PerkinElmer)
e. 15-mL and 50-mL centrifuge tubes, sterile,	Thermo Fisher	05-539-12 and 05-
polypropylene	Scientific	539-8
f. 5-mL Macro Centrifuge Tube, Sterile	Argos	T2076S-CA
g. Wellpro tips (for serial dilution and reagent	Wellpro	T00802
transfer, optional)	Group
h. GripTip Pipette Tips, 5 racks of 96 tips, 300	Integra	6535
μL low retention sterile tips with filter
i. Automation Friendly Reagent Reservoirs,	Integra	6348 (300 mL), 6305
150 mL & 300 mL sterile polypropylene reservoirs		(bases), 6306 (lids),
with base and lid		6338 (150 mL), 6301
		(bases), 6302 (lids)
j. Vi-Cell sample vials	Beckman	723908-D
	Coulter
k. Pipette-Aid	Drummond	Any Suitable
l. Serological pipettes, sterile	Any suitable	NA
m. Single-channel, 8- and 12-channel pipettes	Any suitable	N/A
n. Pipette tips with filter	Any suitable	N/A
o. Solution reservoirs (100 ml), sterile	Any suitable	N/A
p. 50-mL dark polypropylene tubes (for light-	CellTreat	229434 (CellTreat
sensitive reagents)	Scientific or	Scientific) or TB5000
	Argos	(Argos)
q. Black 96-well assay plate lids (for light-	Corning	3935
protection of assay plates)

Reagents used in the examples are provided in Table 3 below.

TABLE 3
Reagent Name	Manufacturer	Cat. No.
a. A549 working cell bank (WCB),	Merck	Merck WCB, do not
currently qualified lot		substitute
b. RSV-NLucP (RSV-NLP) virus bank	Merck	Merck virus bank, do not
(WVS), currently qualified lot		substitute
c. F-12K Nutrient Mixture	Corning, or	10-025-CV (Corning),
	equivalent from	30-2004 (ATCC), 21127-
	ATCC, Gibco	022 (Gibco)
d. FBS-HI (Fetal Bovine Serum, heat	Sigma or	F-4135 (Sigma) or
inactivated)	equivalent from	SH30071.03 (Hyclone)
	Hyclone
e. Penicillin-Streptomycin solution	Gibco	15140-122
(10,000 U/mL)
f. Cell Dissociation Buffer	Corning Inc or	25056CI (Corning) or
(CellStripper or Cell Dissociation Buffer-	Gibco	13151-014 (Gibco)
Enzyme Free)
g. PBS, 1X, pH 7.1-7.4	Thermo Fisher	10010-031
	Scientific
h. Trypan Blue 0.4% solution	Thermo Fisher	15250-061
	Scientific
i. Nano-Glo ™ Luciferase Assay	Promega	N1150
System

Standards and Controls

Standards and controls used in the following examples are described as follows. Currently qualified Antibody A Reference Standard lot was used in each assay. Assay control material was drug substance material which is different from the Reference Standard lot. Currently qualified Assay Control lot was used for each dilution plate to ensure the method and related equipment functions as expected.

This method is suitable for testing anti-RSV antibodies such as Antibody A drug substance and drug product release and stability samples, as well as for miscellaneous samples supporting various studies, for example, extended characterization, analytical comparability etc.

Example 1

Generation of RSV-NLucP (RSV-NLP)

The respiratory syncytial virus P3 (RSV-P3) was first used to carry a reporter gene, placing nanoluciferase and luciferase in between the coding sequences for the M and G protein of RSV-P3. However, expression of the reporters was not successful. Insertion of the nanoluciferase reporter gene between the coding sequences for P and M genes of RSV strain A2 was successful, as described below.

The nucleic acid sequence encoding respiratory syncytial virus A2 (RSV-A2) was cloned into a pSMART vector using multiple portions. A gene cassette comprising rpsL flanked by Mlu restriction sites (rpsL-neo selection/counter-selection cassette; pRedET (tc^R) plasmid; GeneBridges, Heidelberg, Germany) was cloned into the intergenic sequence between P and M using the following recombineering technique.

Amplification of rpsL Cassette

The rpsL-neo cassette (SEQ ID NO: 11) was PCR amplified using the forward and reverse primers of SEQ ID NO: 12 and 13. PCR amplification with Q5 polymerase (New England BioLabs, Ipswich, MA, USA) used 1-2 ng rpsL-neo pRedET (tc^R) plasmid template isolated from Dam+E. Coli with the following PCR cycle: 94° C. for 15 sec., 60° C. for 30 sec., 72° C. for 1 min., for 30 cycles. To remove plasmid template methylated by the Dam+E. Coli, 1-2 μl DpnI was added per 25 μl reaction, mixed, and incubated at 37° C. for 1 hour. DpnI cleaves the methylated the rpsL plasmid template, but does not cleave the unmethylated PCR products. The DpnI-digested PCR product was gel-purified. A strong PCR band was purified, and eluted in 50 μl ddH₂O, and then used to transform electrocompetent SW102 cells (2.5 μl, approximately 10-30 ng).

Insertion of rpsL-neo Cassette into RSV-A2 BAC (Making A2-rpsL BAC)

An overnight culture of SW102 cells containing the RSV-A2 BAC was inoculated in 5 ml LB and chloramphenicol and incubated at 32° C. A portion (500 μl) of the overnight SW102 culture containing the target BAC was diluted in 25 ml LB with chloramphenicol (12.5 mg/ml) in a 50 ml baffled conical flask and incubated at 32° C. in a shaking water bath to an OD600 of approximately. (0.55-0.6), for 3-4 hours. Another portion (10 ml) of the inoculated cells was transferred to another baffled 50 ml conical flask and heat-shocked at 42° C. for exactly 15 minutes in a shaking waterbath. The remaining culture was left at 32° C. as the un-induced control. After 15 minutes, induced and un-induced samples were briefly cooled in an ice/waterbath slurry and then transferred to two 15 ml tubes and pelleted using a centrifuge spun at 5000 RPM at 0° C. for 5 minutes. The supernatant was removed, and the pellet was resuspended in 1 ml ice-cold ddH2O by gently swirling the tubes in the ice/waterbath slurry. When resuspended, another 9 ml ice-cold ddH2O was added and the samples were re-pelleted. The resuspension/re-pelleting step was repeated. After the second washing and centrifugation step, all supernatant was removed by inverting the tubes, and the pellet (approximately 50 ml each) was kept on ice until electroporated with PCR product.

The electrocompetent SW102 cells were then transformed. Each electroporation used 25 μl cells in a 0.1 cm cuvette (BioRad) at 25 mF, 1.75 kV, and 200 ohms. After electroporation of the PCR product, the bacteria were recovered in 1 ml LB (15 ml tube) for 1 hour in a 32° C. shaking waterbath. Following the recovery period, the bacteria were placed on an LB/chloramphenicol+kanamycin agar plate and maintained at 32° C. A clone was picked and checked using colony PCR.

PCR Amplification of Nanoluciferase Cassette (GS-Nlucp-GE Cassette)

A nanoluciferase gene cassette comprising nanoluciferase gene flanked by the gene start (GS) and gene end (GE) sequences of RSV NS2 (GS-Nlucp-GE; SEQ ID NO: 15) was PCR amplified using the forward and reverse primers of SEQ ID NO: 19 and 20.

PCR amplification with Q5 polymerase (New England BioLabs, Ipswich MA) used 1-2 ng template (a GS-Nlucp-GE plasmid) isolated from Dam+E. Coli with the following PCR cycle: 94° C. for 15 sec., 60° C. for 30 sec., 72° C. for 1 min., for 30 cycles. To remove plasmid template methylated by the Dam+E. Coli, 1-2 μl DpnI was added per 25 μl reaction, mixed, and incubated at 37° C. for 1 hour. DpnI cleaves the methylated the rpsL plasmid template, but does not cleave the unmethylated PCR products. The DpnI-digested PCR product was purified using Qiagen quick spin columns.

Transform Bacteria with A2-rpsL BAC

Cells containing the A2-rpsL BAC were cultured (20 ml), and DNA was isolated from the cells and eluted in 30 ul water. The A2-rpsL BAC (1 μl) was then used to transform replication competent E. coli via electroporation (25 μl of cells in a 0.1 cm cuvette at 25 mF, 1.75 kV and 200 ohms). After electroporation, the bacteria were recovered in 1 ml LB (15 ml tube) for 1 hour in a 32° C. shaking waterbath. After the recovery period the bacteria were placed on an LB/chloramphenicol+kanamycin agar plate and maintained at 32° C.

Gibson Assembly to Replace the rpsL Cassette with GS-Nlucp-GE Cassette

The A2-rpsL BAC-transformed bacteria were cultured to a volume of 200 ml in LB+chloramphenicol+0.01% arabinose. The A2-rpsL BAC was then purified using the BACMAX™ DNA Purification Kit (Cambio, Cambridge, UK).

The purified A2-rpsL BAC was digested using 1 μl DpnI to digest the template DNA at 37° C. for 1 hour, and the PCR products were cleaned using Qiaquick PCR Purification Kit (Qiagen, Hilden, Germany).

Assembly product was mixed using purified A2-rpsL BAC fragment (Mlu I digestion; 9 ul), GS-Nlucp-GE cassette PCR product (1:20 dilution; 1 μl), ddH₂O (8 μl), and Gibson Assembly Master Mix (2×) (10 μl). Assembly product was warmed to 50° C. for 15 minutes, then chilled.

Competent cells were thawed on ice, and 20 μl of the chilled assembly product were added to the competent cells, mixing gently. The mixture was placed on ice for 30 minutes, then heat shocked at 42° C. for 30 seconds. Tubes were then transferred to ice for 2 minutes, and 500 μl of room-temperature SOC media was added to the tube. The tube was incubated at 37° C. for 60 minutes, then shaken vigorously (250 rpm) or rotated. The cells (100 μl) were then spread on warmed selection plates (37° C.) and incubated overnight at 37° C.

Two colonies from each of two positive plates were picked, inoculated in 5 ml LB with chloramphenicol and grown overnight with shaking. Colony PCR was used to amplify product using forward and reverse primers for nanolucP (SEQ ID NO: 19 and 20, respectively). PCR cycle initial denaturation 98° C. for 2 minutes, followed by 35 Cycles of : 98° C. for 10 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 30 seconds. Final extension was 72° C. for 2 minutes.

The resulting plasmid pSMART RS A2 NLucP is listed in SEQ ID NO: 21.

Example 2

Viral Rescue

Initial Cell Culture

For transfections, BSR T7/5 cells from “donor” cultures were subpassed into 6 well plates to be 80-90% confluent at time of transfection. One 25 cm² culture was used to prepare one 6 well plate (1:2.5 passage ratio) (or 2 ml of 4e5 cells/ml).

Six-well cell culture plates were prepared for transfection from 25 cm² donor cultures. Growth medium was aspirated from the flasks, and then 0.25 mL of warm trypsin-EDTA was added per 25 cm² flask. Flasks were shaken to distribute the trypsin-EDTA and incubated at 37° C. for 5 to 10 minutes. When cells started to dislodge from the flask, 12 mL of medium was added to each flask, and the cells were suspended by pipetting. Two mL of the cell suspension were added to each well in the 6-well cell culture plates. The cell culture plates were incubated at 37° C. in the tissue culture incubator overnight.

Reagent Preparation for Transfection

Lipofectamine and plasmid/helper plasmid were mixed in a 3:1 ratio (μL/μg), as well as lipofectamine-only and wild type virus-only controls. Each component was diluted with Opti-MEM to make 100 μL of lipofectamine/plasmid/helper plasmid (3:1), lipofectamine-only control, and wild-type virus-only control. Each dilution was incubated at room temperature for 5 minutes.

After incubating the diluted lipofectamine/plasmid/helper plasmid and controls, the following six components were combined in one vial, mixed gently. and incubate the transfection mixture at room temperature for 20 minutes (transfection mixtures should be 600 μL total, Opti-MEM, Lipofectin, and DNA).

The following amounts of each component was used per transfection:

i. RSV antigenome 0.8 μg (8 μL of 0.1 μg/μL)+92 μL Opti-MEM

ii. pCDNA3-L, L protein 0.2 μg (2 μL of 0.1 μg/μL)+98 μL Opti-MEM

iii. pCDNA3-N, N protein 0.4 μg (4 μL of 0.1 μg/μL)+96 μL Opti-MEM

iv. pCDNA3-P, P protein 0.4 μg (4 μL of 0.1 μg/μL)+96 μL Opti-MEM

v. pCDNA3-M2-1, M2-1 protein 0.4 μg (4 μL of 0.1 μg/μL)+96 μL Opti-MEM

vi. Lipofectamine 2000 6.6 μL+93.4 μL Opti-MEM

Cell Transfection

The media from the B SR T7/5 cell culture wells was aspirated, and cells were washed twice with 1 mL warm Opti-MEM. After the final wash was aspirated, 600 μL of the transfection mixtures was added to each culture well, and the cultured cells were incubated for 2 hours at room temperature on a shaker/rocker plate set at low speed. After 2 hours, an additional 600 μL warm Opti-MEM was added per well and incubated at 37° C. overnight (8-12 hours).

After incubation, the transfection mixture was aspirated from the wells and discarded, and each well was washed once with 1 mL warm sterile PBS and replaced with 2 mL of warm growth medium per well. Cultured cells were then incubated at 37° C. overnight.

The cells were then sub-passaged at a 1:3 surface area ratio into 25 cm² flasks using the trypsin-EDTA procedure described above. If the cell morphology appeared weak, the surface area ratio was decreased accordingly up to an even 1:1 ratio (surface area of each well in the 6 well plates is 10 cm²). Cells remained in DMEM with 3% FBS while recovering recovery. Flasks were monitored for cytopathic effect (CPE) and sub-passaged at a 1:3 surface area ratio into new 25 cm² flasks as needed (approximately every 48 hours). CPE appeared first as mini-syncytia and then grew into rounded up clumps of cells. When CPE was evident throughout the flask, the cells were scraped into the growth media, aliquoted into cryovials, and frozen at −80° C. or colder.

Example 3

Preparation of In-Lab Reagents

a) A549 Growth Medium Preparation

TABLE 4
A549 Growth Medium (Note: volumes may
be scaled up or down proportionally)
	Final		Storage
	Conc.	Amount	Temp.	Storage
Reagent	(%)	(mL)	(° C.)	Time
F-12K nutrient mixture	89	445	5 ± 3	1 month
FBS HI*	10	50
Pen/Strep (10,000 U/mL)	1	5
*FBS is aliquoted for single use and stored at −20° C.

Table 4 components were added to make up 500 mL of A549 cell growth medium. A549 cell growth medium was sterile filtered through a 0.2 micron 500-mL (or other appropriate volume) PES filtering unit.

b) A549 Infection Medium Preparation

TABLE 5
A549 Infection Medium (Note: volumes may
be scaled up or down proportionally)
	Final		Storage
	Conc.	Amount	Temp.	Storage
Reagent	(%)	(mL)	(° C.)	Time
F-12K nutrient mixture	97	485	5 ± 3	1 month
FBS HI*	2	10
Pen/Strep (10,000 U/mL)	1	5
*FBS is aliquoted for single use and stored at −20° C.

Table 5 components were added to make up 500 mL of A549 infection medium. A549 infection medium was sterile-filtered through a 0.2 micron 500-mL (or other appropriate volume) PES filtering unit.

Example 4

Procedures for Preparing Testing Samples and Measuring Read-Out Signals

Procedures for preparing testing samples and measuring read-out signals are shown in FIG. 1. Detailed procedures are provided below.

Day 1: Cell Seeding

A549 growth medium was equilibrated to RT, about 30 minutes. Growth medium was aspirated, and cells were washed with 15 mL of PBS with rocking to ensure the entire monolayer was coated. After the PBS was aspirated, 5 mL of cell dissociation buffer solution was added with 15 rocking to ensure the entire monolayer is coated. Each flask was incubated with humidity at 37±1° C., 5±1% CO₂ until cells dislodged from flask (about 10-15 min). Each flask was tapped to disperse the cells.

20 mL of A549 growth medium was added to each flask, and all flask contents were transferred into a single sterile container. The cell suspension was mixed with a serological pipette by slowly pipetting 4-5 times until no clumps were visible. Cells were then counted, either manually or using Vi-Cell. Cell were then suspended in A549 growth medium at 2×10⁵ cells/mL in a sterile PET bottle or equivalent container.

The 100 μl cell suspension was dispensed into 96-well plates (100 μl per well, about 20,000 cells per well), and incubated on a flat surface at RT for 35±5 minutes to ensure that cells homogenously settle dat the bottom of the 96-well plates. Seeded plates were incubated in humidity at 37±1° C., 5±1% CO₂ incubator for 20-26 h.

Day 2: Sample Dilution, Virus Neutralization and Infection

Preparations of Standards, Assay Control and Test Samples

All dilutions are prepared in BSC class II. Thoroughly mix each dilution by vortexing or pipetting during preparation. For each assay plate, dilutions of Antibody A are performed in singleton and tested in duplicate (i.e., each assay dilution is loaded into 2 wells) on each plate. Volumes in the following tables can be scaled up proportionally.

Antibody A Reference Standard was diluted in A549 Infection Medium to 16 μg/mL.

TABLE 6
Example dilution scheme (if Reference
Standard concentration is 101.4 mg/mL):
	Starting	Volume of	Volume of	Target
	Conc.	Starting	Infection	Conc.
Step	(μg/mL)	Material (μL)	Medium (μL)	(μg/mL)
1	101,400	20	2515	800
2	800	40	1960	16

Dilute Antibody A Assay Control was diluted in A549 Infection Medium to 16 μg/mL.

TABLE 7
Example dilution scheme (if Assay Control
concentration is 101.4 mg/mL)
	Starting	Volume of	Volume of	Target
	Conc.	Starting	Infection	Conc.
Step	(μg/mL)	Material (μL)	Medium (μL)	(μg/mL)
1	101,400	20	2515	800
2	800	40	1960	16

TABLE 8
Example dilution scheme (if Assay
Control concentration is 150 mg/mL)
	Starting	Volume of	Volume of	Target
	Conc.	Starting	Infection	Conc.
Step	(μg/mL)	Material (μL)	Medium (μL)	(μg/mL)
1	150,000	20	2480	1200
2	1200	40	2960	16

Antibody A Test Sample(s) was diluted in A549 Infection Medium to 16 μg/mL.

TABLE 9
Example dilution scheme (if Test Sample
concentration is 100 mg/mL)
	Starting	Volume of	Volume of	Target
	Conc.	Starting	Infection	Conc.
Step	(μg/mL)	Material (μL)	Medium (μL)	(μg/mL)
1	100,000	20	2480	800
2	800	40	1960	16

TABLE 10
Example dilution scheme (if Test Sample
concentration is 150 mg/mL)
	Starting	Volume of	Volume of	Target
	Conc.	Starting	Infection	Conc.
Step	(μg/mL)	Material (μL)	Medium (μL)	(μg/mL)
1	150,000	20	2480	1200
2	1200	40	2960	16

Set Up Dilution Plates

Dilution plate maps for plates 1-3 are shown in FIGS. 2A, 2B and 2C. Detailed procedures are described below.

Infection medium (90 μL) was dispensed into wells of a dilution plate, followed by 120 μL of dilutions of Antibody A Reference Standard, Assay Control, and Testing Sample in duplicate into dilution plate wells according to the dilution Plate Maps shown in FIGS. 2A-2C. Four-fold serial dilutions were performed by transfering 30 μL to next column, from column 1 through column 11.

Virus Neutralization

Frozen RSV-NLucP was quickly thawed in 37° C. water bath with gentle agitation until a small portion of ice remained. Once virus was completely thawed, the vial surface was cleaned and wiped with approved disinfectant and the vial was transferred to a Biological Safety Cabinet (BSC).

The desired volume of virus dilution was prepared as shown in table 11.

TABLE 11
Example virus dilution for 1-3 assay plates
			Virus	Infection	Total
Number			stock	Medium	volume
of plates	Virus	Dilution	(mL)	(mL)	(mL)
1	RSV-NLucP WVS	1:40	0.4	15.6	16
2	RSV-NLucP WVS	1:40	0.6	23.4	24
3	RSV-NLucP WVS	1:40	0.9	35.1	36
Note:
Virus dilution factor may be adjusted as an outcome of new lot qualification.

Diluted virus (90 μl) was added to each well of column 11-1 by column (from column 11 backward to column 1) of each plate. Infection medium (90 μl) was added to column-12 row B-D wells (Cell Control). Diluted virus (90 μl) was added to column-12 row E-G wells (Virus Control). Infection medium (180 μl) was added to row A and row H wells. The plate was incubated with 150-200 rpm shaking for 2 minutes, then incubated with humidity at 37±1° C., 5±1% CO₂ incubator for 35±5 min (Neutralization). The virus neutralization time of each plate was documented.

Infection

After incubation, cell plates were examined under a microscope to ensure that the cells were 70%-100% confluent and free of any contamination. The cell plates were equilibrated to RT for 10-15 min in a BSC, and then culture medium was decanted into a waste container and gently tapped upside-down on an absorbent towel to remove residual medium. Virus-sample mixture (90 μL) was transferred to the corresponding wells in each cell culture plate by row (Row A through Row H). Plates were then incubated in humidity at 37±1° C., 5±1% CO₂ for 21±1 h. The inoculation time of each plate was documented.

Day 3: Detection of Infected Cell Plates with Nano-Glo® Reagent

After 21±1 h post infection, the cell plates were equilibrated for 10-15 min at RT. Nano-Glo® reagent was prepared and reconstituted according to manufacturer instructions, and then added to the wells of the cell plate. Cell plates were protected from light, and incubated on a shaker (200 rpm) for 10-15 min at RT, document

Following incubation, plates were read using a Spectramax M5 or M5e Plate Reader as listed in Table 12 below.

TABLE 12
Settings for SpectraMax M5 or M5e Plate Reader Instrument
Parameter              Setting
Read Mode              Luminescence
                       Integration: 500 ms
                       Top read
Read Type              Endpoint
Wavelengths            Lm1
                       Em: All
Assay Plate Type Plate 96-well Polystyrene Clear Flat Bottom Microplate
Read Area              Read entire plate
Shake                  OFF
Calibrate              On
Carriage Speed         Normal
Read Priority          Column Priority

System Suitability Requirements

System suitability was evaluated in each cell plate of the assay run. The Reference Standard and the Assay Control should meet all Assay Acceptance Criteria in Table 13. Failing system suitability requirements disqualified all sample testing results on that cell plate.

Data Acquisition

Raw data containing relative luminescence units (RLU) counts was captured using SoftMax Pro software.

Calculations

The potency of the Testing Sample relative to the Reference Standard (relative potency) was calculated instead of relying on four-parameter logistic regression. Calculating relative potency allows for reducing the amount of duplicated wells and fewer antibody concentrations to be tested. In turn, this allows room in the assay wells for adding not only the reference standard wells, but also Assay Control wells to confirm assay quality.

The mean relative luminescent units (RLU) were plotted as a function of Antibody A concentration to generate sigmoidal dose-response curves. The data were fit to four-parameter logistic (4 PL) equation:

$y=D+\frac{A-D}{1+{\left(\frac{x}{C}\right)}^B}$

where y is the mean luminescence signal, D is the lower asymptote, A is the upper asymptote, x is the Antibody A concentration (ng/mL), C is the inflection point or IC₅₀ (ng/mL), and B is the slope parameter of the 4PL curve fit. Raw data was fitted for each individual test article (i.e., Reference, Control, and Samples) independently of each other (e.g. an unrestricted fit), from which parameters A, B, C, and D for each individual dose-response curve were calculated and recorded.

Determined A and D parameters were used to calculate the relative percentage difference of the upper asymptotes (% A Difference) and the relative percentage difference of the lower asymptotes (% D difference) between Reference and Test samples (or Control) using the following formulas:

$\%A\mathrm{Difference}=❘\frac{❘A_{\mathrm{Reference}}-A_{\mathrm{Sample}\left(\mathrm{or}\mathrm{Control}\right)}❘}{A_{\mathrm{Reference}}-D_{\mathrm{Reference}}}❘\times 100$ $\%D\mathrm{Difference}=❘\frac{❘D_{\mathrm{Reference}}-D_{\mathrm{Sample}\left(\mathrm{or}\mathrm{Control}\right)}❘}{A_{\mathrm{Reference}}-D_{\mathrm{Reference}}}❘\times 100$

Determined B (Slope) parameters were used in calculation of Slopes Ratio using the following equation:

$\mathrm{Slope}\mathrm{Ratio}=\frac{B_{\mathrm{Reference}}}{B_{\mathrm{Sample}\left(\mathrm{or}\mathrm{Control}\right)}}$

A and D parameters calculated for Reference Standard are used to calculate the ratio of the asymptotes as follows:

$\mathrm{Asymptotes}{\mathrm{Ratio}}_{\mathrm{Reference}}=\frac{A_{\mathrm{Reference}}}{D_{\mathrm{Reference}}}$

Sum of Squared Errors (SSE) evaluates goodness of fit statistics and measures the total deviation of the observed response values (experimental) from the fitted data (predicted).

$\mathrm{SSE}=\sum _{i=1}^n{\left(y_i-f_i\right)}^2$

where n is the number of observations (equals to 11 as per the total number of concentration points), y_i is the observed data value and f_i is the predicted value from the fit. SSE was determined based on normalized data to account for possible differences in absolute signal intensity between different instruments.

After the unrestricted analysis, the raw data were fit to a restricted 4PL fit, where Reference curve was analyzed as paired with each individual sample (or control) on the plate, i.e., Reference vs. Sample 1, or Reference vs. Sample 2, or Reference vs. Sample 3, or Reference vs. Assay Control. Both curves in the pair were fit to the same parameter A, the same parameter D, and the same parameter B, allowing only parameter C to fluctuate for the best fit (e.g. Parallel Line Analysis; PLA). PLA results were presented for each pair on a separate graph.

By fitting a pair of curves using PLA analysis, potency of test sample or control was calculated using following equation:

$\mathrm{Relative}\mathrm{Potency}{\left(\%\right)}_{Sample\left(\mathrm{or}\mathrm{Control}\right)}=\frac{C_{\mathrm{Reference}}}{C_{\mathrm{Sample}\left(\mathrm{or}\mathrm{Control}\right)}}\times 100$

Reportable Potency result as geometric mean (GeoMean) of three relative potency values was calculated using the following equation:

GeoMean (%)=10^{Mean(Log10 potency)}

where Mean (Log10 potency) is the average of decimal logarithm values of three Relative Potency values calculated in Section 6.2.0.

Variability of the Reportable Potency as geometric standard deviation (% GSD) of three relative potency values using the following equation:

% GSD=100×(10^{St.Dev.(Log10 potency)}−1)

where St.Dev.(Log10 potency) is standard deviation of decimal logarithm values of three Relative Potency values. If not all 3 plates passed acceptance criteria, the sample testing was repeated according to the Valid Test Acceptance Criteria (see below).

Valid Test Acceptance Criteria

TABLE 13
Assay Acceptance Criteria (for each assay plate):
Assay Article ID	Assay Acceptance Parameter	Acceptance Criteria
Reference	Asymptotes ratio	≥3.0
Standard	SSE of normalized curve fit	≤0.5
Assay Control	SSE of normalized curve fit	≤0.5
	% A Difference vs.	≤25%
	Reference Standard Curve
	% D Difference vs.	≤15%
	Reference Standard Curve
	Ratio of Slopes vs.	0.5-2.0
	Reference Standard Curve
	Relative potency (%)	50-200

Assay Control material is described above (Standards and Controls). Acceptance criteria may be reassessed later when more data are available. If any of the assay acceptance criteria failed, all sample data from that plate was rejected, and the failed plate was re-measured.

TABLE 14
Sample Acceptance Criteria (for each assay plate)
Test Article ID	Sample Acceptance Parameter	Acceptance Criteria
Test Sample	SSE of normalized curve fit	≤0.5
	% A Difference vs.	≤25%
	Reference Standard Curve
	% D Difference vs.	≤15%
	Reference Standard Curve
	Ratio of Slopes vs.	0.5-2.0
	Reference Standard Curve
	Relative potency (%)	Per Qualified Range

If any of the sample acceptance criteria failed for a particular sample, that sample data was rejected, and the failed sample was re-measured. During the re-measured test, sample position on repeat plate was the same as on the original plate.

Acceptance Criteria for Validity of Geometrical Mean Relative Potency (Reportable Result)

The % GSD of the three valid relative potency values was ≤50%. If % GSD>50%, all of the data was evaluated, and results were compared between all three valid plates. If, upon evaluation, atypical results were identified, those plate(s) were invalidated and repeated while maintaining the same plate number(s) and sample positions as in the original plate(s). If no atypical behavior was identified in any of the plates, all three plates were invalidated and repeated.

Reporting Test Results

If all valid acceptance criteria listed in Tables 13 and 14 were successfully met, and the % GSD of the three valid relative potency values was ≤50%, the Geometric Mean of three relative potency values rounded to the same number of decimals was reported.

Relative potency of a test sample is expressed as a percentage relative to concurrently analyzed Antibody A reference standard. The reportable value of potency of Antibody A test sample was calculated as geometric mean of three relative potency determinations obtained from three independently handled assay plates.

TABLE 15
Table of Sequences
SEQ
ID
NO:	Description	Sequence
1	Antibody A	DSAMS
	HC-CDR1
2	Antibody A	FIKSKTYGGTKEYAASVKG
	HC-CDR2
3	Antibody A	GAPYGGNSDYYYGLDV
	HC-CDR3
4	Antibody A	RTSQDVRGALA
	LC-CDR1
5	Antibody A	DASSLET
	LC-CDR2
6	Antibody A	QQFLDFPFT
	LC-CDR3
7	Antibody A VH	EVQLVESGGGLVRPGRSLRLSCTVSGFSFDDSAMSWVRQAPGKGLE
		WISFIKSKTYGGTKEYAASVKGRFTISRDDSKNIAYLQMNSLKTED
		TAVYYCTRGAPYGGNSDYYYGLDVWGQGTTVTVSS
8	Antibody A VL	EVQLVESGGGLVRPGRSLRLSCTVSGFSFDDSAMSWVRQAPGKGLE
		WISFIKSKTYGGTKEYAASVKGRFTISRDDSKNIAYLQMNSLKTED
		TAVYYCTRGAPYGGNSDYYYGLDVWGQGTTVIVSS
9	Antibody A	EVQLVESGGGLVRPGRSLRLSCTVSGFSFDDSAMSWVRQAPGKGLE
	Heavy chain	WISFIKSKTYGGTKEYAASVKGRFTISRDDSKNIAYLQMNSLKTED
		TAVYYCTRGAPYGGNSDYYYGLDVWGQGTTVTVSSASTKGPSVFPL
		APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
		QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
		DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDV
		SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
		WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL
		TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
		FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
10	Antibody A	DIQMTQSPSSLSASVGDRVTITCRTSQDVRGALAWYQQKPGKAPKL
	Light chain	LIFDASSLETGVPSRFSGSGSGTVFTLTISSLOPEDFAAYYCQQFL
		DFPFTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN
		FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA
		DYEKHKVYACEVTHQGLSSPVTKSENRGEC
11	rpsL-neo	ggcctggtgatgatggcgggatcgttgtatatttcttgacaccttt
	cassette	tcggcatcgccctaaaattcggcgtcctcatattgtgtgaggacgt
	Promoter: nt	tttattacgtgtttacgaagcaaaagctaaaaccaggagctattta
	1-138	atggcaacagttaaccagctggtacgcaaaccacgtgctcgcaaag
	rpsL CDS:	ttgcgaaaagcaacgtgcctgcgctggaagcatgcccgcaaaaacg
	139-513	tggcgtatgtactcgtgtatatactaccactcctaaaaaaccgaac
	Neomycin/	tccgcgctgcgtaaagtatgccgtgttcgtctgactaacggtttcg
	kanamycin CDS:	aagtgacttcctacatcggtggtgaaggtcacaacctgcaggagca
	525-2638	ctccgtgatcctgatccgtggcggtcgtgttaaagacctcccgggt
		gttcgttaccacaccgtacgtggtgcgcttgactgctccggcgtta
		aagaccgtaagcaggctcgttccaagtatggcgtgaagcgtcctaa
		ggcttaaggaggacaatcatgattgaacaagatggattgcacgcag
		gttctccggccgcttgggtggagaggctattcggctatgactgggc
		acaacagacaatcggctgctctgatgccgccgtgttccggctgtca
		gcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtg
		ccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggc
		cacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaa
		gcgggaagggactggctgctattgggcgaagtgccggggcaggatc
		tcctgtcatctcaccttgctcctgccgagaaagtatccatcatggc
		tgatgcaatgcggcggctgcatacgcttgatccggctacctgccca
		ttcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga
		tggaagccggtcttgtcgatcaggatgatctggacgaagagcatca
		ggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatg
		cccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgc
		cgaatatcatggtggaaaatggccgcttttctggattcatcgactg
		tggccggctgggtgtggcggaccgctatcaggacatagcgttggct
		acccgtgatattgctgaagagcttggcggcgaatgggctgaccgct
		tcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgc
		cttctatcgccttcttgacgagttcttctga
12	Forward	CTTCGTGAAGCTTGTTCACGTATGTTTCCATATTTGCCCCACCCTA
	primer for rpsL	cgcgtGGCCTGGTGATGATGGCGGGATCG
	cassette
13	Reverse primer	CTAACCACCCGGAAAAAATCTATAATATAGTTACAAAAAAAGGAAa
	for rpsL	cgcgTCAGAAGAACTCGTCAAGAAGG
	cassette
14	RSV A2	ACTTGCATAAACCAAAAAAATGGGGCAAATAAGAATTTGATAAGTA
	NS1 CDS: nt	CCACTTAAATTTAACTCCCTTGGTTAGAGATGGGCAGCAATTCATT
	76-492	GAGTATGATAAAAGTTAGATTACAAAATTTGTTTGACAATGATGAA
	NS2 CDS: nt	GTAGCATTGTTAAAAATAACATGCTATACTGATAAATTAATACATT
	9605-76	TAACTAACGCTTTGGCTAAGGCAGTGATACATACAATCAAATTGAA
	N CDS: nt	TGGCATTGTGTTTGTGCATGTTATTACAAGTAGTGATATTTGCCCT
	1117-2289	AATAATAATATTGTAGTAAAATCCAATTTCACAACAATGCCAGTAC
	P CDS:	TACAAAATGGAGGTTATATATGGGAAATGATGGAATTAACACATTG
	2323-3045	CTCTCAACCTAATGGTCTACTAGATGACAATTGTGAAATTAAATTC
	M CDS:	TCCAAAAAACTAAGTGATTCAACAATGACCAATTATATGAATCAAT
	3238-4005	TATCTGAATTACTTGGATTTGATCTTAATCCATAAATTATAATTAA
	SH CDS:	TATCAACTAGCAAATCAATGTCACTAACACCATTAGTTAATATAAA
	4280-4471	ACTTAACAGAAGACAAAAATGGGGCAAATAAATCAATTCAGCCAAC
	G CDS:	CCAACCATGGACACAACCCACAATGATAATACACCACAAAGACTGA
	4665-5558	TGATCACAGACATGAGACCGTTGTCACTTGAGACCATAATAACATC
	F CDS:	ACTAACCAGAGACATCATAACACACAAATTTATATACTTGATAAAT
	5638-7359	CATGAATGCATAGTGAGAAAACTTGATGAAAAACAGGCCACATTTA
	M2-1 CDS:	CATTCCTGGTCAACTATGAAATGAAACTATTACACAAAGTAGGAAG
	7583-8164	CACTAAATATAAAAAATATACTGAATACAACACAAAATATGGCACT
	M2-2 CDS:	TTCCCTATGCCAATATTCATCAATCATGATGGGTTCTTAGAATGCA
	8136-8405	TTGGCATTAAGCCTACAAAGCATACTCCCATAATATACAAGTATGA
	L CDS:	TCTCAATCCATAAATTTCAACACAATATTCACACAATCTAAAACAA
	8475-14,969	CAACTCTATGCATAACTATACTCCATAGTCCAGATGGAGCCTGAAA
		ATTATAGTAATTTAAAATTAAGGAGAGATATAAGATAGAAGATGGG
		GCAAATACAAAGATGGCTCTTAGCAAAGTCAAGTTGAATGATACAC
		TCAACAAAGATCAACTTCTGTCATCCAGCAAATACACCATCCAACG
		GAGCACAGGAGATAGTATTGATACTCCTAATTATGATGTGCAGAAA
		CACATCAATAAGTTATGTGGCATGTTATTAATCACAGAAGATGCTA
		ATCATAAATTCACTGGGTTAATAGGTATGTTATATGCGATGTCTAG
		GTTAGGAAGAGAAGACACCATAAAAATACTCAGAGATGCGGGATAT
		CATGTAAAAGCAAATGGAGTAGATGTAACAACACATCGTCAAGACA
		TTAATGGAAAAGAAATGAAATTTGAAGTGTTAACATTGGCAAGCTT
		AACAACTGAAATTCAAATCAACATTGAGATAGAATCTAGAAAATCC
		TACAAAAAAATGCTAAAAGAAATGGGAGAGGTAGCTCCAGAATACA
		GGCATGACTCTCCTGATTGTGGGATGATAATATTATGTATAGCAGC
		ATTAGTAATAACTAAATTAGCAGCAGGGGACAGATCTGGTCTTACA
		GCCGTGATTAGGAGAGCTAATAATGTCCTAAAAAATGAAATGAAAC
		GTTACAAAGGCTTACTACCCAAGGACATAGCCAACAGCTTCTATGA
		AGTGTTTGAAAAACATCCCCACTTTATAGATGTTTTTGTTCATTTT
		GGTATAGCACAATCTTCTACCAGAGGTGGCAGTAGAGTTGAAGGGA
		TTTTTGCAGGATTGTTTATGAATGCCTATGGTGCAGGGCAAGTGAT
		GTTACGGTGGGGAGTCTTAGCAAAATCAGTTAAAAATATTATGTTA
		GGACATGCTAGTGTGCAAGCAGAAATGGAACAAGTTGTTGAGGTTT
		ATGAATATGCCCAAAAATTGGGTGGTGAAGCAGGATTCTACCATAT
		ATTGAACAACCCAAAAGCATCATTATTATCTTTGACTCAATTTCCT
		CACTTCTCCAGTGTAGTATTAGGCAATGCTGCTGGCCTAGGCATAA
		TGGGAGAGTACAGAGGTACACCGAGGAATCAAGATCTATATGATGC
		AGCAAAGGCATATGCTGAACAACTCAAAGAAAATGGTGTGATTAAC
		TACAGTGTACTAGACTTGACAGCAGAAGAACTAGAGGCTATCAAAC
		ATCAGCTTAATCCAAAAGATAATGATGTAGAGCTTTGAGTTAATAA
		AAAATGGGGCAAATAAATCATCATGGAAAAGTTTGCTCCTGAATTC
		CATGGAGAAGATGCAAACAACAGGGCTACTAAATTCCTAGAATCAA
		TAAAGGGCAAATTCACATCACCCAAAGATCCCAAGAAAAAAGATAG
		TATCATATCTGTCAACTCAATAGATATAGAAGTAACCAAAGAAAGC
		CCTATAACATCAAATTCAACTATTATCAACCCAACAAATGAGACAG
		ATGATACTGCAGGGAACAAGCCCAATTATCAAAGAAAACCTCTAGT
		AAGTTTCAAAGAAGACCCTACACCAAGTGATAATCCCTTTTCTAAA
		CTATACAAAGAAACCATAGAAACATTTGATAACAATGAAGAAGAAT
		CCAGCTATTCATACGAAGAAATAAATGATCAGACAAACGATAATAT
		AACAGCAAGATTAGATAGGATTGATGAAAAATTAAGTGAAATACTA
		GGAATGCTTCACACATTAGTAGTGGCAAGTGCAGGACCTACATCTG
		CTCGGGATGGTATAAGAGATGCCATGATTGGTTTAAGAGAAGAAAT
		GATAGAAAAAATCAGAACTGAAGCATTAATGACCAATGACAGATTA
		GAAGCTATGGCAAGACTCAGGAATGAGGAAAGTGAAAAGATGGCAA
		AAGACACATCAGATGAAGTGTCTCTCAATCCAACATCAGAGAAATT
		GAACAACCTATTGGAAGGGAATGATAGTGACAATGATCTATCACTT
		GAAGATTTCTGATTAGTTACCACTCTTCACATCAACACACAATACC
		AACAGAAGACCAACAAACTAACCAACCCAATCATCCAACCAAACAT
		CCATCCGCCAATCAGCCAAACAGCCAACAAAACAACCAGCCAATCC
		AAAACTAACCACCCGGAAAAAATCTATAATATAGTTACAAAAAAAG
		GAAAGGGTGGGGCAAATATGGAAACATACGTGAACAAGCTTCACGA
		AGGCTCCACATACACAGCTGCTGTTCAATACAATGTCTTAGAAAAA
		GACGATGACCCTGCATCACTTACAATATGGGTGCCCATGTTCCAAT
		CATCTATGCCAGCAGATTTACTTATAAAAGAACTAGCTAATGTCAA
		CATACTAGTGAAACAAATATCCACACCCAAGGGACCTTCACTAAGA
		GTCATGATAAACTCAAGAAGTGCAGTGCTAGCACAAATGCCCAGCA
		AATTTACCATATGCGCTAATGTGTCCTTGGATGAAAGAAGCAAACT
		AGCATATGATGTAACCACACCCTGTGAAATCAAGGCATGTAGTCTA
		ACATGCCTAAAATCAAAAAATATGTTGACTACAGTTAAAGATCTCA
		CTATGAAGACACTCAACCCTACACATGATATTATTGCTTTATGTGA
		ATTTGAAAACATAGTAACATCAAAAAAAGTCATAATACCAACATAC
		CTAAGATCCATCAGTGTCAGAAATAAAGATCTGAACACACTTGAAA
		ATATAACAACCACTGAATTCAAAAATGCTATCACAAATGCAAAAAT
		CATCCCTTACTCAGGATTACTATTAGTCATCACAGTGACTGACAAC
		AAAGGAGCATTCAAATACATAAAGCCACAAAGTCAATTCATAGTAG
		ATCTTGGAGCTTACCTAGAAAAAGAAAGTATATATTATGTTACCAC
		AAATTGGAAGCACACAGCTACACGATTTGCAATCAAACCCATGGAA
		GATTAACCTTTTTCCTCTACATCAGTGTGTTAATTCATACAAACTT
		TCTACCTACATTCTTCACTTCACCATCACAATCACAAACACTCTGT
		GGTTCAACCAATCAAACAAAACTTATCTGAAGTCCCAGATCATCCC
		AAGTCATTGTTTATCAGATCTAGTACTCAAATAAGTTAATAAAAAA
		TATACACATGGGGCAAATAATCATTGGAGGAAATCCAACTAATCAC
		AATATCTGTTAACATAGACAAGTCCACACACCATACAGAATCAACC
		AATGGAAAATACATCCATAACAATAGAATTCTCAAGCAAATTCTGG
		CCTTACTTTACACTAATACACATGATCACAACAATAATCTCTTTGC
		TAATCATAATCTCCATCATGATTGCAATACTAAACAAACTTTGTGA
		ATATAACGTATTCCATAACAAAACCTTTGAGTTACCAAGAGCTCGA
		GTCAACACATAGCATTCATCAATCCAACAGCCCAAAACAGTAACCT
		TGCATTTAAAAATGAACAACCCCTACCTCTTTACAACACCTCATTA
		ACATCCCACCATGCAAACCACTATCCATACTATAAAGTAGTTAATT
		AAAAATAGTCATAACAATGAACTAGGATATCAAGACTAACAATAAC
		ATTGGGGCAAATGCAAACATGTCCAAAAACAAGGACCAACGCACCG
		CTAAGACATTAGAAAGGACCTGGGACACTCTCAATCATTTATTATT
		CATATCATCGTGCTTATATAAGTTAAATCTTAAATCTGTAGCACAA
		ATCACATTATCCATTCTGGCAATGATAATCTCAACTTCACTTATAA
		TTGCAGCCATCATATTCATAGCCTCGGCAAACCACAAAGTCACACC
		AACAACTGCAATCATACAAGATGCAACAAGCCAGATCAAGAACACA
		ACCCCAACATACCTCACCCAGAATCCTCAGCTTGGAATCAGTCCCT
		CTAATCCGTCTGAAATTACATCACAAATCACCACCATACTAGCTTC
		AACAACACCAGGAGTCAAGTCAACCCTGCAATCCACAACAGTCAAG
		ACCAAAAACACAACAACAACTCAAACACAACCCAGCAAGCCCACCA
		CAAAACAACGCCAAAACAAACCACCAAGCAAACCCAATAATGATTT
		TCACTTTGAAGTGTTCAACTTTGTACCCTGCAGCATATGCAGCAAC
		AATCCAACCTGCTGGGCTATCTGCAAAAGAATACCAAACAAAAAAC
		CAGGAAAGAAAACCACTACCAAGCCCACAAAAAAACCAACCCTCAA
		GACAACCAAAAAAGATCCCAAACCTCAAACCACTAAATCAAAGGAA
		GTACCCACCACCAAGCCCACAGAAGAGCCAACCATCAACACCACCA
		AAACAAACATCATAACTACACTACTCACCTCCAACACCACAGGAAA
		TCCAGAACTCACAAGTCAAATGGAAACCTTCCACTCAACTTCCTCC
		GAAGGCAATCCAAGCCCTTCTCAAGTCTCTACAACATCCGAGTACC
		CATCACAACCTTCATCTCCACCCAACACACCACGCCAGTAGTTACT
		TAAAAACATATTATCACAAAAAGCCATGACCAACTTAAACAGAATC
		AAAATAAACTCTGGGGCAAATAACAATGGAGTTGCTAATCCTCAAA
		GCAAATGCAATTACCACAATCCTCACTGCAGTCACATTTTGTTTTG
		CTTCTGGTCAAAACATCACTGAAGAATTTTATCAATCAACATGCAG
		TGCAGTTAGCAAAGGCTATCTTAGTGCTCTGAGAACTGGTTGGTAT
		ACCAGTGTTATAACTATAGAATTAAGTAATATCAAGGAAAATAAGT
		GTAATGGAACAGATGCTAAGGTAAAATTGATAAAACAAGAATTAGA
		TAAATATAAAAATGCTGTAACAGAATTGCAGTTGCTCATGCAAAGC
		ACACCACCAACAAACAATCGAGCCAGAAGAGAACTACCAAGGTTTA
		TGAATTATACACTCAACAATGCCAAAAAAACCAATGTAACATTAAG
		CAAGAAAAGGAAAAGAAGATTTCTTGTTTTTTTGTTAGGTGTTGGA
		TCTGCAATCGCCAGTGGCGTTGCTGTATCTAAGGTCCTGCACCTAG
		AAGGGGAAGTGAACAAGATCAAAAGTGCTCTACTATCCACAAACAA
		GGCTCTAGTCAGCTTATCAAATGGAGTTAGTGTCTTAACCAGCAAA
		GTGTTAGACCTCAAAAACTATATAGATAAACAATTGTTACCTATTG
		TGAACAAGCAAAGCTGCAGCATATCAAATATAGAAACTGTGATAGA
		GTTCCAACAAAAGAACAACAGACTACTAGAGATTACCAGGGAATTT
		AGTGTTAATGCAGGTGTAACTACACCTGTAAGCACTTACATGTTAA
		CTAATAGTGAATTATTGTCATTAATCAATGATATGCCTATAACAAA
		TGATCAGAAAAAGTTAATGTCCAACAATGTTCAAATAGTTAGACAG
		CAAAGTTACTCTATCATGTCCATAATAAAAGAGGAAGTCTTAGCAT
		ATGTAGTACAATTACCACTATATGGTGTTATAGATACACCCTGTTG
		GAAACTACACACATCCCCTCTATGTACAACCAACACAAAAGAAGGG
		TCCAACATCTGTTTAACAAGAACTGACAGAGGATGGTACTGTGACA
		ATGCAGGATCAGTATCTTTCTTCCCACAAGCTGAAACATGTAAAGT
		TCAATCAAATCGAGTATTTTGTGACACAATGAACAGTTTAACATTA
		CCAAGTGAAATAAATCTCTGCAATGTTGACATATTCAACCCCAAAT
		ATGATTGTAAAATTATGACTTCAAAAACAGATGTAAGCAGCTCCGT
		TATCACATCTCTAGGAGCCATTGTGTCATGCTATGGCAAAACTAAA
		TGTACAGCATCCAATAAAAATCGTGGAATCATAAAGACATTTTCTA
		ACGGGTGCGATTATGTATCAAATAAAGGGATGGACACTGTGTCTGT
		AGGTAACACATTATATTATGTAAATAAGCAAGAAGGTAAAAGTCTC
		TATGTAAAAGGTGAACCAATAATAAATTTCTATGACCCATTAGTAT
		TCCCCTCTGATGAATTTGATGCATCAATATCTCAAGTCAACGAGAA
		GATTAACCAGAGCCTAGCATTTATTCGTAAATCCGATGAATTATTA
		CATAATGTAAATGCTGGTAAATCCACCACAAATATCATGATAACTA
		CTATAATTATAGTGATTATAGTAATATTGTTATCATTAATTGCTGT
		TGGACTGCTCTTATACTGTAAGGCCAGAAGCACACCAGTCACACTA
		AGCAAAGATCAACTGAGTGGTATAAATAATATTGCATTTAGTAACT
		AAATAAAAATAGCACCTAATCATGTTCTTACAATGGTTTACTATCT
		GCTCATAGACAACCCATCTGTCATTGGATTTTCTTAAAATCTGAAC
		TTCATCGAAACTCTCATCTATAAACCATCTCACTTACACTATTTAA
		GTAGATTCCTAGTTTATAGTTATATAAAACACAATTGAATGCCAGA
		TTAACTTACCATCTGTAAAAATGAAAACTGGGGCAAATATGTCACG
		AAGGAATCCTTGCAAATTTGAAATTCGAGGTCATTGCTTAAATGGT
		AAGAGGTGTCATTTTAGTCATAATTATTTTGAATGGCCACCCCATG
		CACTGCTTGTAAGACAAAACTTTATGTTAAACAGAATACTTAAGTC
		TATGGATAAAAGTATAGATACCTTATCAGAAATAAGTGGAGCTGCA
		GAGTTGGACAGAACAGAAGAGTATGCTCTTGGTGTAGTTGGAGTGC
		TAGAGAGTTATATAGGATCAATAAACAATATAACTAAACAATCAGC
		ATGTGTTGCCATGAGCAAACTCCTCACTGAACTCAATAGTGATGAT
		ATCAAAAAGCTGAGGGACAATGAAGAGCTAAATTCACCCAAGATAA
		GAGTGTACAATACTGTCATATCATATATTGAAAGCAACAGGAAAAA
		CAATAAACAAACTATCCATCTGTTAAAAAGATTGCCAGCAGACGTA
		TTGAAGAAAACCATCAAAAACACATTGGATATCCATAAGAGCATAA
		CCATCAACAACCCAAAAGAATCAACTGTTAGTGATACAAATGACCA
		TGCCAAAAATAATGATACTACCTGACAAATATCCTTGTAGTATAAC
		TTCCATACTAATAACAAGTAGATGTAGAGTTACTATGTATAATCAA
		AAGAACACACTATATTTCAATCAAAACAACCCAAATAACCATATGT
		ACTCACCGAATCAAACATTCAATGAAATCCATTGGACCTCTCAAGA
		ATTGATTGACACAATTCAAATTTTTCTACAACATCTAGGTATTATT
		GAGGATATATATACAATATATATATTAGTGTCATAACACTCAATTC
		TAACACTCACCACATCGTTACATTATTAATTCAAACAATTCAAGTT
		GTGGGACAAAATGGATCCCATTATTAATGGAAATTCTGCTAATGTT
		TATCTAACCGATAGTTATTTAAAAGGTGTTATCTCTTTCTCAGAGT
		GTAATGCTTTAGGAAGTTACATATTCAATGGTCCTTATCTCAAAAA
		TGATTATACCAACTTAATTAGTAGACAAAATCCATTAATAGAACAC
		ATGAATCTAAAGAAACTAAATATAACACAGTCCTTAATATCTAAGT
		ATCATAAAGGTGAAATAAAATTAGAAGAACCTACTTATTTTCAGTC
		ATTACTTATGACATACAAGAGTATGACCTCGTCAGAACAGATTGCT
		ACCACTAATTTACTTAAAAAGATAATAAGAAGAGCTATAGAAATAA
		GTGATGTCAAAGTCTATGCTATATTGAATAAACTAGGGCTTAAAGA
		AAAGGACAAGATTAAATCCAACAATGGACAAGATGAAGACAACTCA
		GTTATTACGACCATAATCAAAGATGATATACTTTCAGCTGTTAAAG
		ATAATCAATCTCATCTTAAAGCAGACAAAAATCACTCTACAAAACA
		AAAAGACACAATCAAAACAACACTCTTGAAGAAATTGATGTGTTCA
		ATGCAACATCCTCCATCATGGTTAATACATTGGTTTAACTTATACA
		CAAAATTAAACAACATATTAACACAGTATCGATCAAATGAGGTAAA
		AAACCATGGGTTTACATTGATAGATAATCAAACTCTTAGTGGATTT
		CAATTTATTTTGAACCAATATGGTTGTATAGTTTATCATAAGGAAC
		TCAAAAGAATTACTGTGACAACCTATAATCAATTCTTGACATGGAA
		AGATATTAGCCTTAGTAGATTAAATGTTTGTTTAATTACATGGATT
		AGTAACTGCTTGAACACATTAAATAAAAGCTTAGGCTTAAGATGCG
		GATTCAATAATGTTATCTTGACACAACTATTCCTTTATGGAGATTG
		TATACTAAAGCTATTTCACAATGAGGGGTTCTACATAATAAAAGAG
		GTAGAGGGATTTATTATGTCTCTAATTTTAAATATAACAGAAGAAG
		ATCAATTCAGAAAACGATTTTATAATAGTATGCTCAACAACATCAC
		AGATGCTGCTAATAAAGCTCAGAAAAATCTGCTATCAAGAGTATGT
		CATACATTATTAGATAAGACAGTGTCCGATAATATAATAAATGGCA
		GATGGATAATTCTATTAAGTAAGTTCCTTAAATTAATTAAGCTTGC
		AGGTGACAATAACCTTAACAATCTGAGTGAACTATATTTTTTGTTC
		AGAATATTTGGACACCCAATGGTAGATGAAAGACAAGCCATGGATG
		CTGTTAAAATTAATTGCAATGAGACCAAATTTTACTTGTTAAGCAG
		TCTGAGTATGTTAAGAGGTGCCTTTATATATAGAATTATAAAAGGG
		TTTGTAAATAATTACAACAGATGGCCTACTTTAAGAAATGCTATTG
		TTTTACCCTTAAGATGGTTAACTTACTATAAACTAAACACTTATCC
		TTCTTTGTTGGAACTTACAGAAAGAGATTTGATTGTGTTATCAGGA
		CTACGTTTCTATCGTGAGTTTCGGTTGCCTAAAAAAGTGGATCTTG
		AAATGATTATAAATGATAAAGCTATATCACCTCCTAAAAATTTGAT
		ATGGACTAGTTTCCCTAGAAATTACATGCCATCACACATACAAAAC
		TATATAGAACATGAAAAATTAAAATTTTCCGAGAGTGATAAATCAA
		GAAGAGTATTAGAGTATTATTTAAGAGATAACAAATTCAATGAATG
		TGATTTATACAACTGTGTAGTTAATCAAAGTTATCTCAACAACCCT
		AATCATGTGGTATCATTGACAGGCAAAGAAAGAGAACTCAGTGTAG
		GTAGAATGTTTGCAATGCAACCGGGAATGTTCAGACAGGTTCAAAT
		ATTGGCAGAGAAAATGATAGCTGAAAACATTTTACAATTCTTTCCT
		GAAAGTCTTACAAGATATGGTGATCTAGAACTACAAAAAATATTAG
		AATTGAAAGCAGGAATAAGTAACAAATCAAATCGCTACAATGATAA
		TTACAACAATTACATTAGTAAGTGCTCTATCATCACAGATCTCAGC
		AAATTCAATCAAGCATTTCGATATGAAACGTCATGTATTTGTAGTG
		ATGTGCTGGATGAACTGCATGGTGTACAATCTCTATTTTCCTGGTT
		ACATTTAACTATTCCTCATGTCACAATAATATGCACATATAGGCAT
		GCACCCCCCTATATAGGAGATCATATTGTAGATCTTAACAATGTAG
		ATGAACAAAGTGGATTATATAGATATCACATGGGTGGCATCGAAGG
		GTGGTGTCAAAAACTGTGGACCATAGAAGCTATATCACTATTGGAT
		CTAATATCTCTCAAAGGGAAATTCTCAATTACTGCTTTAATTAATG
		GTGACAATCAATCAATAGATATAAGCAAACCAATCAGACTCATGGA
		AGGTCAAACTCATGCTCAAGCAGATTATTTGCTAGCATTAAATAGC
		CTTAAATTACTGTATAAAGAGTATGCAGGCATAGGCCACAAATTAA
		AAGGAACTGAGACTTATATATCACGAGATATGCAATTTATGAGTAA
		AACAATTCAACATAACGGTGTATATTACCCAGCTAGTATAAAGAAA
		GTCCTAAGAGTGGGACCGTGGATAAACACTATACTTGATGATTTCA
		AAGTGAGTCTAGAATCTATAGGTAGTTTGACACAAGAATTAGAATA
		TAGAGGTGAAAGTCTATTATGCAGTTTAATATTTAGAAATGTATGG
		TTATATAATCAGATTGCTCTACAATTAAAAAATCATGCATTATGTA
		ACAATAAACTATATTTGGACATATTAAAGGTTCTGAAACACTTAAA
		AACCTTTTTTAATCTTGATAATATTGATACAGCATTAACATTGTAT
		ATGAATTTACCCATGTTATTTGGTGGTGGTGATCCCAACTTGTTAT
		ATCGAAGTTTCTATAGAAGAACTCCTGACTTCCTCACAGAGGCTAT
		AGTTCACTCTGTGTTCATACTTAGTTATTATACAAACCATGACTTA
		AAAGATAAACTTCAAGATCTGTCAGATGATAGATTGAATAAGTTCT
		TAACATGCATAATCACGTTTGACAAAAACCCTAATGCTGAATTCGT
		AACATTGATGAGAGATCCTCAAGCTTTAGGGTCTGAGAGACAAGCT
		AAAATTACTAGCGAAATCAATAGACTGGCAGTTACAGAGGTTTTGA
		GTACAGCTCCAAACAAAATATTCTCCAAAAGTGCACAACATTATAC
		TACTACAGAGATAGATCTAAATGATATTATGCAAAATATAGAACCT
		ACATATCCTCATGGGCTAAGAGTTGTTTATGAAAGTTTACCCTTTT
		ATAAAGCAGAGAAAATAGTAAATCTTATATCAGGTACAAAATCTAT
		AACTAACATACTGGAAAAAACTTCTGCCATAGACTTAACAGATATT
		GATAGAGCCACTGAGATGATGAGGAAAAACATAACTTTGCTTATAA
		GGATACTTCCATTGGATTGTAACAGAGATAAAAGAGAGATATTGAG
		TATGGAAAACCTAAGTATTACTGAATTAAGCAAATATGTTAGGGAA
		AGATCTTGGTCTTTATCCAATATAGTTGGTGTTACATCACCCAGTA
		TCATGTATACAATGGACATCAAATATACTACAAGCACTATATCTAG
		TGGCATAATTATAGAGAAATATAATGTTAACAGTTTAACACGTGGT
		GAGAGAGGACCCACTAAACCATGGGTTGGTTCATCTACACAAGAGA
		AAAAAACAATGCCAGTTTATAATAGACAAGTCTTAACCAAAAAACA
		GAGAGATCAAATAGATCTATTAGCAAAATTGGATTGGGTGTATGCA
		TCTATAGATAACAAGGATGAATTCATGGAAGAACTCAGCATAGGAA
		CCCTTGGGTTAACATATGAAAAGGCCAAGAAATTATTTCCACAATA
		TTTAAGTGTCAATTATTTGCATCGCCTTACAGTCAGTAGTAGACCA
		TGTGAATTCCCTGCATCAATACCAGCTTATAGAACAACAAATTATC
		ACTTTGACACTAGCCCTATTAATCGCATATTAACAGAAAAGTATGG
		TGATGAAGATATTGACATAGTATTCCAAAACTGTATAAGCTTTGGC
		CTTAGTTTAATGTCAGTAGTAGAACAATTTACTAATGTATGTCCTA
		ACAGAATTATTCTCATACCTAAGCTTAATGAGATACATTTGATGAA
		ACCTCCCATATTCACAGGTGATGTTGATATTCACAAGTTAAAACAA
		GTGATACAAAAACAGCATATGTTTTTACCAGACAAAATAAGTTTGA
		CTCAATATGTGGAATTATTCTTAAGTAATAAAACACTCAAATCTGG
		ATCTCATGTTAATTCTAATTTAATATTGGCACATAAAATATCTGAC
		TATTTTCATAATACTTACATTTTAAGTACTAATTTAGCTGGACATT
		GGATTCTGATTATACAACTTATGAAAGATTCTAAAGGTATTTTTGA
		AAAAGATTGGGGAGAGGGATATATAACTGATCATATGTTTATTAAT
		TTGAAAGTTTTCTTCAATGCTTATAAGACCTATCTCTTGTGTTTTC
		ATAAAGGTTATGGCAAAGCAAAGCTGGAGTGTGATATGAACACTTC
		AGATCTTCTATGTGTATTGGAATTAATAGACAGTAGTTATTGGAAG
		TCTATGTCTAAGGTATTTTTAGAACAAAAAGTTATCAAATACATTC
		TTAGCCAAGATGCAAGTTTACATAGAGTAAAAGGATGTCATAGCTT
		CAAATTATGGTTTCTTAAACGTCTTAATGTAGCAGAATTCACAGTT
		TGCCCTTGGGTTGTTAACATAGATTATCATCCAACACATATGAAAG
		CAATATTAACTTATATAGATCTTGTTAGAATGGGATTGATAAATAT
		AGATAGAATACACATTAAAAATAAACACAAATTCAATGATGAATTT
		TATACTTCTAATCTCTTCTACATTAATTATAACTTCTCAGATAATA
		CTCATCTATTAACTAAACATATAAGGATTGCTAATTCTGAATTAGA
		AAATAATTACAACAAATTATATCATCCTACACCAGAAACCCTAGAG
		AATATACTAGCCAATCCGATTAAAAGTAATGACAAAAAGACACTGA
		ATGACTATTGTATAGGTAAAAATGTTGACTCAATAATGTTACCATT
		GTTATCTAATAAGAAGCTTATTAAATCGTCTGCAATGATTAGAACC
		AATTACAGCAAACAAGATTTGTATAATTTATTCCCTATGGTTGTGA
		TTGATAGAATTATAGATCATTCAGGCAATACAGCCAAATCCAACCA
		ACTTTACACTACTACTTCCCACCAAATATCTTTAGTCCACAATAGC
		ACATCACTTTACTGCATGCTTCCTTGGCATCATATTAATAGATTCA
		ATTTTGTATTTAGTTCTACAGGTTGTAAAATTAGTATAGAGTATAT
		TTTAAAAGATCTTAAAATTAAAGATCCCAATTGTATAGCATTCATA
		GGTGAAGGAGCAGGGAATTTATTATTGCGTACAGTAGTGGAACTTC
		ATCCTGACATAAGATATATTTACAGAAGTCTGAAAGATTGCAATGA
		TCATAGTTTACCTATTGAGTTTTTAAGGCTGTACAATGGACATATC
		AACATTGATTATGGTGAAAATTTGACCATTCCTGCTACAGATGCAA
		CCAACAACATTCATTGGTCTTATTTACATATAAAGTTTGCTGAACC
		TATCAGTCTTTTTGTCTGTGATGCCGAATTGTCTGTAACAGTCAAC
		TGGAGTAAAATTATAATAGAATGGAGCAAGCATGTAAGAAAGTGCA
		AGTACTGTTCCTCAGTTAATAAATGTATGTTAATAGTAAAATATCA
		TGCTCAAGATGATATTGATTTCAAATTAGACAATATAACTATATTA
		AAAACTTATGTATGCTTAGGCAGTAAGTTAAAGGGATCGGAGGTTT
		ACTTAGTCCTTACAATAGGTCCTGCGAATATATTCCCAGTATTTAA
		TGTAGTACAAAATGCTAAATTGATACTATCAAGAACCAAAAATTTC
		ATCATGCCTAAGAAAGCTGATAAAGAGTCTATTGATGCAAATATTA
		AAAGTTTGATACCCTTTCTTTGTTACCCTATAACAAAAAAAGGAAT
		TAATACTGCATTGTCAAAACTAAAGAGTGTTGTTAGTGGAGATATA
		CTATCATATTCTATAGCTGGACGTAATGAAGTTTTCAGCAATAAAC
		TTATAAATCATAAGCATATGAACATCTTAAAATGGTTCAATCATGT
		TTTAAATTTCAGATCAACAGAACTAAACTATAACCATTTATATATG
		GTAGAATCTACATATCCTTACCTAAGTGAATTGTTAAACAGCTTGA
		CAACCAATGAACTTAAAAAACTGATTAAAATCACAGGTAGTCTGTT
		ATACAACTTTCATAATGAATAATGAATAAAGATCTTATAATAAAAA
		TTCCCATAGCTATACACTAACACTGTATTCAATTATAGTTATTAAA
		AATTAAAAATCATATAATTTTTTAAATAACTTTTAGTGAACTAATC
		CTAAAGTTATCATTTTAATCTTGGAGGAATAAATTTAAACCCTAAT
		CTAATTGGTTTATATGTGTATTAACTAAATTACGAGATATTAGTTT
		TTGACACTTTTTTTCTCGT
15	GS-NlucP-GE	GGGGCAAATAAATCAATGGTCTTCACACTCGAAGATTTCGTTGGGG
	GS: nt 1-15	ACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACA
	NlucP: nt	GGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACT
	616-54	CCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCG
	GE: nt	ACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAAT
	655-671	GGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGAT
		CATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACG
		GGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGG
		CATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTG
		TGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACG
		GCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCG
		GCTGTGCGAACGCATTCTGGCGAATTCTCACGGCTTTCCGCCTGAG
		GTTGAAGAGCAAGCCGCCGGTACATTGCCTATGTCCTGCGCACAAG
		AAAGCGGTATGGACCGGCACCCAGCCGCTTGTGCTTCAGCTCGCAT
		CAACGTCTAAATTATAGTAATTTAAAA
16	RSV gene start	GGGGCAAATAAATCA
	sequence
17	RSV gene end	ATTATAGTAATTTAAAA
	sequence
18	NanolucP gene	ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAG
		CCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAG
		TTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATT
		GTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCA
		TCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAA
		AATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG
		ATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACA
		TGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGA
		CGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAA
		ATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCC
		GAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCAT
		TCTGGCGAATTCTCACGGCTTTCCGCCTGAGGTTGAAGAGCAAGCC
		GCCGGTACATTGCCTATGTCCTGCGCACAAGAAAGCGGTATGGACC
		GGCACCCAGCCGCTTGTGCTTCAGCTCGCATCAACGTCTAA
19	Forward	TATAGTTACAAAAAAAGGAAGGGGCAAATAAATCAATGGTCTTCAC
	primer for	ACTCGAAGATT
	NanolucP
20	Reverse primer	TTCCATATTTGCCCCACCCTTTTTAAATTACTATAATTTAGACGTT
	for NanolucP	GATGCGAGCTG
21	pSMART RS	GGCGCGCCTAATACGACTCACTATAGGGACGGGAAAAAATGCGTAC
	A2 NLucP	AACAAACTTGCATAAACCAAAAAAATGGGGCAAATAAGAATTTGAT
	NS1 CDS: nt	AAGTACCACTTAAATTTAACTCCCTTGGTTAGAGATGGGCAGCAAT
	127-543	TCATTGAGTATGATAAAAGTTAGATTACAAAATTTGTTTGACAATG
	NS2 CDS: nt	ATGAAGTAGCATTGTTAAAAATAACATGCTATACTGATAAATTAAT
	656-1027	ACATTTAACTAACGCTTTGGCTAAGGCAGTGATACATACAATCAAA
	N CDS: nt	TTGAATGGCATTGTGTTTGTGCATGTTATTACAAGTAGTGATATTT
	1168-2340	GCCCTAATAATAATATTGTAGTAAAATCCAATTTCACAACAATGCC
	P CDS: nt	AGTACTACAAAATGGAGGTTATATATGGGAAATGATGGAATTAACA
	2374-3096	CATTGCTCTCAACCTAATGGTCTACTAGATGACAATTGTGAAATTA
	Intergenic P-M	AATTCTCCAAAAAACTAAGTGATTCAACAATGACCAATTATATGAA
	region: nt	TCAATTATCTGAATTACTTGGATTTGATCTTAATCCATAAATTATA
	3271-3950	ATTAATATCAACTAGCAAATCAATGTCACTAACACCATTAGTTAAT
	GS-NlucP-GE:	ATAAAACTTAACAGAAGACAAAAATGGGGCAAATAAATCAATTCAG
	nt 3275-3945	CCAACCCAACCATGGACACAACCCACAATGATAATACACCACAAAG
	GS: nt	ACTGATGATCACAGACATGAGACCGTTGTCACTTGAGACCATAATA
	33275-289	ACATCACTAACCAGAGACATCATAACACACAAATTTATATACTTGA
	GE: nt	TAAATCATGAATGCATAGTGAGAAAACTTGATGAAAAACAGGCCAC
	3929-3945	ATTTACATTCCTGGTCAACTATGAAATGAAACTATTACACAAAGTA
	nanoLucP	GGAAGCACTAAATATAAAAAATATACTGAATACAACACAAAATATG
	CDS: nt	GCACTTTCCCTATGCCAATATTCATCAATCATGATGGGTTCTTAGA
	3290-3928	ATGCATTGGCATTAAGCCTACAAAGCATACTCCCATAATATACAAG
	M CDS: nt	TATGATCTCAATCCATAAATTTCAACACAATATTCACACAATCTAA
	3960-4727	AACAACAACTCTATGCATAACTATACTCCATAGTCCAGATGGAGCC
	SH CDS: nt	TGAAAATTATAGTAATTTAAAATTAAGGAGAGATATAAGATAGAAG
	5002-5193	ATGGGGCAAATACAAAGATGGCTCTTAGCAAAGTCAAGTTGAATGA
	G CDS: nt	TACACTCAACAAAGATCAACTTCTGTCATCCAGCAAATACACCATC
	5387-6280	CAACGGAGCACAGGAGATAGTATTGATACTCCTAATTATGATGTGC
	F CDS: nt	AGAAACACATCAATAAGTTATGTGGCATGTTATTAATCACAGAAGA
	6360-8081	TGCTAATCATAAATTCACTGGGTTAATAGGTATGTTATATGCGATG
	M2-1 CDS: nt	TCTAGGTTAGGAAGAGAAGACACCATAAAAATACTCAGAGATGCGG
	8305-8886	GATATCATGTAAAAGCAAATGGAGTAGATGTAACAACACATCGTCA
	M2-2 CDS: nt	AGACATTAATGGAAAAGAAATGAAATTTGAAGTGTTAACATTGGCA
	8858-9127	AGCTTAACAACTGAAATTCAAATCAACATTGAGATAGAATCTAGAA
	L CDS: nt	AATCCTACAAAAAAATGCTAAAAGAAATGGGAGAGGTAGCTCCAGA
	9197-15,691	ATACAGGCATGACTCTCCTGATTGTGGGATGATAATATTATGTATA
		GCAGCATTAGTAATAACTAAATTAGCAGCAGGGGACAGATCTGGTC
		TTACAGCCGTGATTAGGAGAGCTAATAATGTCCTAAAAAATGAAAT
		GAAACGTTACAAAGGCTTACTACCCAAGGACATAGCCAACAGCTTC
		TATGAAGTGTTTGAAAAACATCCCCACTTTATAGATGTTTTTGTTC
		ATTTTGGTATAGCACAATCTTCTACCAGAGGTGGCAGTAGAGTTGA
		AGGGATTTTTGCAGGATTGTTTATGAATGCCTATGGTGCAGGGCAA
		GTGATGTTACGGTGGGGAGTCTTAGCAAAATCAGTTAAAAATATTA
		TGTTAGGACATGCTAGTGTGCAAGCAGAAATGGAACAAGTTGTTGA
		GGTTTATGAATATGCCCAAAAATTGGGTGGTGAAGCAGGATTCTAC
		CATATATTGAACAACCCAAAAGCATCATTATTATCTTTGACTCAAT
		TTCCTCACTTCTCCAGTGTAGTATTAGGCAATGCTGCTGGCCTAGG
		CATAATGGGAGAGTACAGAGGTACACCGAGGAATCAAGATCTATAT
		GATGCAGCAAAGGCATATGCTGAACAACTCAAAGAAAATGGTGTGA
		TTAACTACAGTGTACTAGACTTGACAGCAGAAGAACTAGAGGCTAT
		CAAACATCAGCTTAATCCAAAAGATAATGATGTAGAGCTTTGAGTT
		AATAAAAAATGGGGCAAATAAATCATCATGGAAAAGTTTGCTCCTG
		AATTCCATGGAGAAGATGCAAACAACAGGGCTACTAAATTCCTAGA
		ATCAATAAAGGGCAAATTCACATCACCCAAAGATCCCAAGAAAAAA
		GATAGTATCATATCTGTCAACTCAATAGATATAGAAGTAACCAAAG
		AAAGCCCTATAACATCAAATTCAACTATTATCAACCCAACAAATGA
		GACAGATGATACTGCAGGGAACAAGCCCAATTATCAAAGAAAACCT
		CTAGTAAGTTTCAAAGAAGACCCTACACCAAGTGATAATCCCTTTT
		CTAAACTATACAAAGAAACCATAGAAACATTTGATAACAATGAAGA
		AGAATCCAGCTATTCATACGAAGAAATAAATGATCAGACAAACGAT
		AATATAACAGCAAGATTAGATAGGATTGATGAAAAATTAAGTGAAA
		TACTAGGAATGCTTCACACATTAGTAGTGGCAAGTGCAGGACCTAC
		ATCTGCTCGGGATGGTATAAGAGATGCCATGATTGGTTTAAGAGAA
		GAAATGATAGAAAAAATCAGAACTGAAGCATTAATGACCAATGACA
		GATTAGAAGCTATGGCAAGACTCAGGAATGAGGAAAGTGAAAAGAT
		GGCAAAAGACACATCAGATGAAGTGTCTCTCAATCCAACATCAGAG
		AAATTGAACAACCTATTGGAAGGGAATGATAGTGACAATGATCTAT
		CACTTGAAGATTTCTGATTAGTTACCACTCTTCACATCAACACACA
		ATACCAACAGAAGACCAACAAACTAACCAACCCAATCATCCAACCA
		AACATCCATCCGCCAATCAGCCAAACAGCCAACAAAACAACCAGCC
		AATCCAAAACTAACCACCCGGAAAAAATCTATAATATAGTTACAAA
		AAAAGGAAGGGGCAAATAAATCAATGGTCTTCACACTCGAAGATTT
		CGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTC
		CTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGT
		CCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCT
		GAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGC
		GACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTG
		TGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGT
		AATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCG
		TATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAG
		GGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAA
		CCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACC
		GGCTGGCGGCTGTGCGAACGCATTCTGGCGAATTCTCACGGCTTTC
		CGCCTGAGGTTGAAGAGCAAGCCGCCGGTACATTGCCTATGTCCTG
		CGCACAAGAAAGCGGTATGGACCGGCACCCAGCCGCTTGTGCTTCA
		GCTCGCATCAACGTCTAAATTATAGTAATTTAAAAAGGGTGGGGCA
		AATATGGAAACATACGTGAACAAGCTTCACGAAGGCTCCACATACA
		CAGCTGCTGTTCAATACAATGTCTTAGAAAAAGACGATGACCCTGC
		ATCACTTACAATATGGGTGCCCATGTTCCAATCATCTATGCCAGCA
		GATTTACTTATAAAAGAACTAGCTAATGTCAACATACTAGTGAAAC
		AAATATCCACACCCAAGGGACCTTCACTAAGAGTCATGATAAACTC
		AAGAAGTGCAGTGCTAGCACAAATGCCCAGCAAATTTACCATATGC
		GCTAATGTGTCCTTGGATGAAAGAAGCAAACTAGCATATGATGTAA
		CCACACCCTGTGAAATCAAGGCATGTAGTCTAACATGCCTAAAATC
		AAAAAATATGTTGACTACAGTTAAAGATCTCACTATGAAGACACTC
		AACCCTACACATGATATTATTGCTTTATGTGAATTTGAAAACATAG
		TAACATCAAAAAAAGTCATAATACCAACATACCTAAGATCCATCAG
		TGTCAGAAATAAAGATCTGAACACACTTGAAAATATAACAACCACT
		GAATTCAAAAATGCTATCACAAATGCAAAAATCATCCCTTACTCAG
		GATTACTATTAGTCATCACAGTGACTGACAACAAAGGAGCATTCAA
		ATACATAAAGCCACAAAGTCAATTCATAGTAGATCTTGGAGCTTAC
		CTAGAAAAAGAAAGTATATATTATGTTACCACAAATTGGAAGCACA
		CAGCTACACGATTTGCAATCAAACCCATGGAAGATTAACCTTTTTC
		CTCTACATCAGTGTGTTAATTCATACAAACTTTCTACCTACATTCT
		TCACTTCACCATCACAATCACAAACACTCTGTGGTTCAACCAATCA
		AACAAAACTTATCTGAAGTCCCAGATCATCCCAAGTCATTGTTTAT
		CAGATCTAGTACTCAAATAAGTTAATAAAAAATATACACATGGGGC
		AAATAATCATTGGAGGAAATCCAACTAATCACAATATCTGTTAACA
		TAGACAAGTCCACACACCATACAGAATCAACCAATGGAAAATACAT
		CCATAACAATAGAATTCTCAAGCAAATTCTGGCCTTACTTTACACT
		AATACACATGATCACAACAATAATCTCTTTGCTAATCATAATCTCC
		ATCATGATTGCAATACTAAACAAACTTTGTGAATATAACGTATTCC
		ATAACAAAACCTTTGAGTTACCAAGAGCTCGAGTCAACACATAGCA
		TTCATCAATCCAACAGCCCAAAACAGTAACCTTGCATTTAAAAATG
		AACAACCCCTACCTCTTTACAACACCTCATTAACATCCCACCATGC
		AAACCACTATCCATACTATAAAGTAGTTAATTAAAAATAGTCATAA
		CAATGAACTAGGATATCAAGACTAACAATAACATTGGGGCAAATGC
		AAACATGTCCAAAAACAAGGACCAACGCACCGCTAAGACATTAGAA
		AGGACCTGGGACACTCTCAATCATTTATTATTCATATCATCGTGCT
		TATATAAGTTAAATCTTAAATCTGTAGCACAAATCACATTATCCAT
		TCTGGCAATGATAATCTCAACTTCACTTATAATTGCAGCCATCATA
		TTCATAGCCTCGGCAAACCACAAAGTCACACCAACAACTGCAATCA
		TACAAGATGCAACAAGCCAGATCAAGAACACAACCCCAACATACCT
		CACCCAGAATCCTCAGCTTGGAATCAGTCCCTCTAATCCGTCTGAA
		ATTACATCACAAATCACCACCATACTAGCTTCAACAACACCAGGAG
		TCAAGTCAACCCTGCAATCCACAACAGTCAAGACCAAAAACACAAC
		AACAACTCAAACACAACCCAGCAAGCCCACCACAAAACAACGCCAA
		AACAAACCACCAAGCAAACCCAATAATGATTTTCACTTTGAAGTGT
		TCAACTTTGTACCCTGCAGCATATGCAGCAACAATCCAACCTGCTG
		GGCTATCTGCAAAAGAATACCAAACAAAAAACCAGGAAAGAAAACC
		ACTACCAAGCCCACAAAAAAACCAACCCTCAAGACAACCAAAAAAG
		ATCCCAAACCTCAAACCACTAAATCAAAGGAAGTACCCACCACCAA
		GCCCACAGAAGAGCCAACCATCAACACCACCAAAACAAACATCATA
		ACTACACTACTCACCTCCAACACCACAGGAAATCCAGAACTCACAA
		GTCAAATGGAAACCTTCCACTCAACTTCCTCCGAAGGCAATCCAAG
		CCCTTCTCAAGTCTCTACAACATCCGAGTACCCATCACAACCTTCA
		TCTCCACCCAACACACCACGCCAGTAGTTACTTAAAAACATATTAT
		CACAAAAAGCCATGACCAACTTAAACAGAATCAAAATAAACTCTGG
		GGCAAATAACAATGGAGTTGCTAATCCTCAAAGCAAATGCAATTAC
		CACAATCCTCACTGCAGTCACATTTTGTTTTGCTTCTGGTCAAAAC
		ATCACTGAAGAATTTTATCAATCAACATGCAGTGCAGTTAGCAAAG
		GCTATCTTAGTGCTCTGAGAACTGGTTGGTATACCAGTGTTATAAC
		TATAGAATTAAGTAATATCAAGGAAAATAAGTGTAATGGAACAGAT
		GCTAAGGTAAAATTGATAAAACAAGAATTAGATAAATATAAAAATG
		CTGTAACAGAATTGCAGTTGCTCATGCAAAGCACACCACCAACAAA
		CAATCGAGCCAGAAGAGAACTACCAAGGTTTATGAATTATACACTC
		AACAATGCCAAAAAAACCAATGTAACATTAAGCAAGAAAAGGAAAA
		GAAGATTTCTTGTTTTTTTGTTAGGTGTTGGATCTGCAATCGCCAG
		TGGCGTTGCTGTATCTAAGGTCCTGCACCTAGAAGGGGAAGTGAAC
		AAGATCAAAAGTGCTCTACTATCCACAAACAAGGCTCTAGTCAGCT
		TATCAAATGGAGTTAGTGTCTTAACCAGCAAAGTGTTAGACCTCAA
		AAACTATATAGATAAACAATTGTTACCTATTGTGAACAAGCAAAGC
		TGCAGCATATCAAATATAGAAACTGTGATAGAGTTCCAACAAAAGA
		ACAACAGACTACTAGAGATTACCAGGGAATTTAGTGTTAATGCAGG
		TGTAACTACACCTGTAAGCACTTACATGTTAACTAATAGTGAATTA
		TTGTCATTAATCAATGATATGCCTATAACAAATGATCAGAAAAAGT
		TAATGTCCAACAATGTTCAAATAGTTAGACAGCAAAGTTACTCTAT
		CATGTCCATAATAAAAGAGGAAGTCTTAGCATATGTAGTACAATTA
		CCACTATATGGTGTTATAGATACACCCTGTTGGAAACTACACACAT
		CCCCTCTATGTACAACCAACACAAAAGAAGGGTCCAACATCTGTTT
		AACAAGAACTGACAGAGGATGGTACTGTGACAATGCAGGATCAGTA
		TCTTTCTTCCCACAAGCTGAAACATGTAAAGTTCAATCAAATCGAG
		TATTTTGTGACACAATGAACAGTTTAACATTACCAAGTGAAATAAA
		TCTCTGCAATGTTGACATATTCAACCCCAAATATGATTGTAAAATT
		ATGACTTCAAAAACAGATGTAAGCAGCTCCGTTATCACATCTCTAG
		GAGCCATTGTGTCATGCTATGGCAAAACTAAATGTACAGCATCCAA
		TAAAAATCGTGGAATCATAAAGACATTTTCTAACGGGTGCGATTAT
		GTATCAAATAAAGGGATGGACACTGTGTCTGTAGGTAACACATTAT
		ATTATGTAAATAAGCAAGAAGGTAAAAGTCTCTATGTAAAAGGTGA
		ACCAATAATAAATTTCTATGACCCATTAGTATTCCCCTCTGATGAA
		TTTGATGCATCAATATCTCAAGTCAACGAGAAGATTAACCAGAGCC
		TAGCATTTATTCGTAAATCCGATGAATTATTACATAATGTAAATGC
		TGGTAAATCCACCACAAATATCATGATAACTACTATAATTATAGTG
		ATTATAGTAATATTGTTATCATTAATTGCTGTTGGACTGCTCTTAT
		ACTGTAAGGCCAGAAGCACACCAGTCACACTAAGCAAAGATCAACT
		GAGTGGTATAAATAATATTGCATTTAGTAACTAAATAAAAATAGCA
		CCTAATCATGTTCTTACAATGGTTTACTATCTGCTCATAGACAACC
		CATCTGTCATTGGATTTTCTTAAAATCTGAACTTCATCGAAACTCT
		CATCTATAAACCATCTCACTTACACTATTTAAGTAGATTCCTAGTT
		TATAGTTATATAAAACACAATTGAATGCCAGATTAACTTACCATCT
		GTAAAAATGAAAACTGGGGCAAATATGTCACGAAGGAATCCTTGCA
		AATTTGAAATTCGAGGTCATTGCTTAAATGGTAAGAGGTGTCATTT
		TAGTCATAATTATTTTGAATGGCCACCCCATGCACTGCTTGTAAGA
		CAAAACTTTATGTTAAACAGAATACTTAAGTCTATGGATAAAAGTA
		TAGATACCTTATCAGAAATAAGTGGAGCTGCAGAGTTGGACAGAAC
		AGAAGAGTATGCTCTTGGTGTAGTTGGAGTGCTAGAGAGTTATATA
		GGATCAATAAACAATATAACTAAACAATCAGCATGTGTTGCCATGA
		GCAAACTCCTCACTGAACTCAATAGTGATGATATCAAAAAGCTGAG
		GGACAATGAAGAGCTAAATTCACCCAAGATAAGAGTGTACAATACT
		GTCATATCATATATTGAAAGCAACAGGAAAAACAATAAACAAACTA
		TCCATCTGTTAAAAAGATTGCCAGCAGACGTATTGAAGAAAACCAT
		CAAAAACACATTGGATATCCATAAGAGCATAACCATCAACAACCCA
		AAAGAATCAACTGTTAGTGATACAAATGACCATGCCAAAAATAATG
		ATACTACCTGACAAATATCCTTGTAGTATAACTTCCATACTAATAA
		CAAGTAGATGTAGAGTTACTATGTATAATCAAAAGAACACACTATA
		TTTCAATCAAAACAACCCAAATAACCATATGTACTCACCGAATCAA
		ACATTCAATGAAATCCATTGGACCTCTCAAGAATTGATTGACACAA
		TTCAAATTTTTCTACAACATCTAGGTATTATTGAGGATATATATAC
		AATATATATATTAGTGTCATAACACTCAATTCTAACACTCACCACA
		TCGTTACATTATTAATTCAAACAATTCAAGTIGTGGGACAAAATGG
		ATCCCATTATTAATGGAAATTCTGCTAATGTTTATCTAACCGATAG
		TTATTTAAAAGGTGTTATCTCTTTCTCAGAGTGTAATGCTTTAGGA
		AGTTACATATTCAATGGTCCTTATCTCAAAAATGATTATACCAACT
		TAATTAGTAGACAAAATCCATTAATAGAACACATGAATCTAAAGAA
		ACTAAATATAACACAGTCCTTAATATCTAAGTATCATAAAGGTGAA
		ATAAAATTAGAAGAACCTACTTATTTTCAGTCATTACTTATGACAT
		ACAAGAGTATGACCTCGTCAGAACAGATTGCTACCACTAATTTACT
		TAAAAAGATAATAAGAAGAGCTATAGAAATAAGTGATGTCAAAGTC
		TATGCTATATTGAATAAACTAGGGCTTAAAGAAAAGGACAAGATTA
		AATCCAACAATGGACAAGATGAAGACAACTCAGTTATTACGACCAT
		AATCAAAGATGATATACTTTCAGCTGTTAAAGATAATCAATCTCAT
		CTTAAAGCAGACAAAAATCACTCTACAAAACAAAAAGACACAATCA
		AAACAACACTCTTGAAGAAATTGATGTGTTCAATGCAACATCCTCC
		ATCATGGTTAATACATTGGTTTAACTTATACACAAAATTAAACAAC
		ATATTAACACAGTATCGATCAAATGAGGTAAAAAACCATGGGTTTA
		CATTGATAGATAATCAAACTCTTAGTGGATTTCAATTTATTTTGAA
		CCAATATGGTTGTATAGTTTATCATAAGGAACTCAAAAGAATTACT
		GTGACAACCTATAATCAATTCTTGACATGGAAAGATATTAGCCTTA
		GTAGATTAAATGTTTGTTTAATTACATGGATTAGTAACTGCTTGAA
		CACATTAAATAAAAGCTTAGGCTTAAGATGCGGATTCAATAATGTT
		ATCTTGACACAACTATTCCTTTATGGAGATTGTATACTAAAGCTAT
		TTCACAATGAGGGGTTCTACATAATAAAAGAGGTAGAGGGATTTAT
		TATGTCTCTAATTTTAAATATAACAGAAGAAGATCAATTCAGAAAA
		CGATTTTATAATAGTATGCTCAACAACATCACAGATGCTGCTAATA
		AAGCTCAGAAAAATCTGCTATCAAGAGTATGTCATACATTATTAGA
		TAAGACAGTGTCCGATAATATAATAAATGGCAGATGGATAATTCTA
		TTAAGTAAGTTCCTTAAATTAATTAAGCTTGCAGGTGACAATAACC
		TTAACAATCTGAGTGAACTATATTTTTTGTTCAGAATATTTGGACA
		CCCAATGGTAGATGAAAGACAAGCCATGGATGCTGTTAAAATTAAT
		TGCAATGAGACCAAATTTTACTTGTTAAGCAGTCTGAGTATGTTAA
		GAGGTGCCTTTATATATAGAATTATAAAAGGGTTTGTAAATAATTA
		CAACAGATGGCCTACTTTAAGAAATGCTATTGTTTTACCCTTAAGA
		TGGTTAACTTACTATAAACTAAACACTTATCCTTCTTTGTTGGAAC
		TTACAGAAAGAGATTTGATTGTGTTATCAGGACTACGTTTCTATCG
		TGAGTTTCGGTTGCCTAAAAAAGTGGATCTTGAAATGATTATAAAT
		GATAAAGCTATATCACCTCCTAAAAATTTGATATGGACTAGTTTCC
		CTAGAAATTACATGCCATCACACATACAAAACTATATAGAACATGA
		AAAATTAAAATTTTCCGAGAGTGATAAATCAAGAAGAGTATTAGAG
		TATTATTTAAGAGATAACAAATTCAATGAATGTGATTTATACAACT
		GTGTAGTTAATCAAAGTTATCTCAACAACCCTAATCATGTGGTATC
		ATTGACAGGCAAAGAAAGAGAACTCAGTGTAGGTAGAATGTTTGCA
		ATGCAACCGGGAATGTTCAGACAGGTTCAAATATTGGCAGAGAAAA
		TGATAGCTGAAAACATTTTACAATTCTTTCCTGAAAGTCTTACAAG
		ATATGGTGATCTAGAACTACAAAAAATATTAGAATTGAAAGCAGGA
		ATAAGTAACAAATCAAATCGCTACAATGATAATTACAACAATTACA
		TTAGTAAGTGCTCTATCATCACAGATCTCAGCAAATTCAATCAAGC
		ATTTCGATATGAAACGTCATGTATTTGTAGTGATGTGCTGGATGAA
		CTGCATGGTGTACAATCTCTATTTTCCTGGTTACATTTAACTATTC
		CTCATGTCACAATAATATGCACATATAGGCATGCACCCCCCTATAT
		AGGAGATCATATTGTAGATCTTAACAATGTAGATGAACAAAGTGGA
		TTATATAGATATCACATGGGTGGCATCGAAGGGTGGTGTCAAAAAC
		TGTGGACCATAGAAGCTATATCACTATTGGATCTAATATCTCTCAA
		AGGGAAATTCTCAATTACTGCTTTAATTAATGGTGACAATCAATCA
		ATAGATATAAGCAAACCAATCAGACTCATGGAAGGTCAAACTCATG
		CTCAAGCAGATTATTTGCTAGCATTAAATAGCCTTAAATTACTGTA
		TAAAGAGTATGCAGGCATAGGCCACAAATTAAAAGGAACTGAGACT
		TATATATCACGAGATATGCAATTTATGAGTAAAACAATTCAACATA
		ACGGTGTATATTACCCAGCTAGTATAAAGAAAGTCCTAAGAGTGGG
		ACCGTGGATAAACACTATACTTGATGATTTCAAAGTGAGTCTAGAA
		TCTATAGGTAGTTTGACACAAGAATTAGAATATAGAGGTGAAAGTC
		TATTATGCAGTTTAATATTTAGAAATGTATGGTTATATAATCAGAT
		TGCTCTACAATTAAAAAATCATGCATTATGTAACAATAAACTATAT
		TTGGACATATTAAAGGTTCTGAAACACTTAAAAACCTTTTTTAATC
		TTGATAATATTGATACAGCATTAACATTGTATATGAATTTACCCAT
		GTTATTTGGTGGTGGTGATCCCAACTTGTTATATCGAAGTTTCTAT
		AGAAGAACTCCTGACTTCCTCACAGAGGCTATAGTTCACTCTGTGT
		TCATACTTAGTTATTATACAAACCATGACTTAAAAGATAAACTTCA
		AGATCTGTCAGATGATAGATTGAATAAGTTCTTAACATGCATAATC
		ACGTTTGACAAAAACCCTAATGCTGAATTCGTAACATTGATGAGAG
		ATCCTCAAGCTTTAGGGTCTGAGAGACAAGCTAAAATTACTAGCGA
		AATCAATAGACTGGCAGTTACAGAGGTTTTGAGTACAGCTCCAAAC
		AAAATATTCTCCAAAAGTGCACAACATTATACTACTACAGAGATAG
		ATCTAAATGATATTATGCAAAATATAGAACCTACATATCCTCATGG
		GCTAAGAGTTGTTTATGAAAGTTTACCCTTTTATAAAGCAGAGAAA
		ATAGTAAATCTTATATCAGGTACAAAATCTATAACTAACATACTGG
		AAAAAACTTCTGCCATAGACTTAACAGATATTGATAGAGCCACTGA
		GATGATGAGGAAAAACATAACTTTGCTTATAAGGATACTTCCATTG
		GATTGTAACAGAGATAAAAGAGAGATATTGAGTATGGAAAACCTAA
		GTATTACTGAATTAAGCAAATATGTTAGGGAAAGATCTTGGTCTTT
		ATCCAATATAGTTGGTGTTACATCACCCAGTATCATGTATACAATG
		GACATCAAATATACTACAAGCACTATATCTAGTGGCATAATTATAG
		AGAAATATAATGTTAACAGTTTAACACGTGGTGAGAGAGGACCCAC
		TAAACCATGGGTTGGTTCATCTACACAAGAGAAAAAAACAATGCCA
		GTTTATAATAGACAAGTCTTAACCAAAAAACAGAGAGATCAAATAG
		ATCTATTAGCAAAATTGGATTGGGTGTATGCATCTATAGATAACAA
		GGATGAATTCATGGAAGAACTCAGCATAGGAACCCTTGGGTTAACA
		TATGAAAAGGCCAAGAAATTATTTCCACAATATTTAAGTGTCAATT
		ATTTGCATCGCCTTACAGTCAGTAGTAGACCATGTGAATTCCCTGC
		ATCAATACCAGCTTATAGAACAACAAATTATCACTTTGACACTAGC
		CCTATTAATCGCATATTAACAGAAAAGTATGGTGATGAAGATATTG
		ACATAGTATTCCAAAACTGTATAAGCTTTGGCCTTAGTTTAATGTC
		AGTAGTAGAACAATTTACTAATGTATGTCCTAACAGAATTATTCTC
		ATACCTAAGCTTAATGAGATACATTTGATGAAACCTCCCATATTCA
		CAGGTGATGTTGATATTCACAAGTTAAAACAAGTGATACAAAAACA
		GCATATGTTTTTACCAGACAAAATAAGTTTGACTCAATATGTGGAA
		TTATTCTTAAGTAATAAAACACTCAAATCTGGATCTCATGTTAATT
		CTAATTTAATATTGGCACATAAAATATCTGACTATTTTCATAATAC
		TTACATTTTAAGTACTAATTTAGCTGGACATTGGATTCTGATTATA
		CAACTTATGAAAGATTCTAAAGGTATTTTTGAAAAAGATTGGGGAG
		AGGGATATATAACTGATCATATGTTTATTAATTTGAAAGTTTTCTT
		CAATGCTTATAAGACCTATCTCTTGTGTTTTCATAAAGGTTATGGC
		AAAGCAAAGCTGGAGTGTGATATGAACACTTCAGATCTTCTATGTG
		TATTGGAATTAATAGACAGTAGTTATTGGAAGTCTATGTCTAAGGT
		ATTTTTAGAACAAAAAGTTATCAAATACATTCTTAGCCAAGATGCA
		AGTTTACATAGAGTAAAAGGATGTCATAGCTTCAAATTATGGTTTC
		TTAAACGTCTTAATGTAGCAGAATTCACAGTTTGCCCTTGGGTTGT
		TAACATAGATTATCATCCAACACATATGAAAGCAATATTAACTTAT
		ATAGATCTTGTTAGAATGGGATTGATAAATATAGATAGAATACACA
		TTAAAAATAAACACAAATTCAATGATGAATTTTATACTTCTAATCT
		CTTCTACATTAATTATAACTTCTCAGATAATACTCATCTATTAACT
		AAACATATAAGGATTGCTAATTCTGAATTAGAAAATAATTACAACA
		AATTATATCATCCTACACCAGAAACCCTAGAGAATATACTAGCCAA
		TCCGATTAAAAGTAATGACAAAAAGACACTGAATGACTATTGTATA
		GGTAAAAATGTTGACTCAATAATGTTACCATTGTTATCTAATAAGA
		AGCTTATTAAATCGTCTGCAATGATTAGAACCAATTACAGCAAACA
		AGATTTGTATAATTTATTCCCTATGGTTGTGATTGATAGAATTATA
		GATCATTCAGGCAATACAGCCAAATCCAACCAACTTTACACTACTA
		CTTCCCACCAAATATCTTTAGTCCACAATAGCACATCACTTTACTG
		CATGCTTCCTTGGCATCATATTAATAGATTCAATTTTGTATTTAGT
		TCTACAGGTTGTAAAATTAGTATAGAGTATATTTTAAAAGATCTTA
		AAATTAAAGATCCCAATTGTATAGCATTCATAGGTGAAGGAGCAGG
		GAATTTATTATTGCGTACAGTAGTGGAACTTCATCCTGACATAAGA
		TATATTTACAGAAGTCTGAAAGATTGCAATGATCATAGTTTACCTA
		TTGAGTTTTTAAGGCTGTACAATGGACATATCAACATTGATTATGG
		TGAAAATTTGACCATTCCTGCTACAGATGCAACCAACAACATTCAT
		TGGTCTTATTTACATATAAAGTTTGCTGAACCTATCAGTCTTTTTG
		TCTGTGATGCCGAATTGTCTGTAACAGTCAACTGGAGTAAAATTAT
		AATAGAATGGAGCAAGCATGTAAGAAAGTGCAAGTACTGTTCCTCA
		GTTAATAAATGTATGTTAATAGTAAAATATCATGCTCAAGATGATA
		TTGATTTCAAATTAGACAATATAACTATATTAAAAACTTATGTATG
		CTTAGGCAGTAAGTTAAAGGGATCGGAGGTTTACTTAGTCCTTACA
		ATAGGTCCTGCGAATATATTCCCAGTATTTAATGTAGTACAAAATG
		CTAAATTGATACTATCAAGAACCAAAAATTTCATCATGCCTAAGAA
		AGCTGATAAAGAGTCTATTGATGCAAATATTAAAAGTTTGATACCC
		TTTCTTTGTTACCCTATAACAAAAAAAGGAATTAATACTGCATTGT
		CAAAACTAAAGAGTGTTGTTAGTGGAGATATACTATCATATTCTAT
		AGCTGGACGTAATGAAGTTTTCAGCAATAAACTTATAAATCATAAG
		CATATGAACATCTTAAAATGGTTCAATCATGTTTTAAATTTCAGAT
		CAACAGAACTAAACTATAACCATTTATATATGGTAGAATCTACATA
		TCCTTACCTAAGTGAATTGTTAAACAGCTTGACAACCAATGAACTT
		AAAAAACTGATTAAAATCACAGGTAGTCTGTTATACAACTTTCATA
		ATGAATAATGAATAAAGATCTTATAATAAAAATTCCCATAGCTATA
		CACTAACACTGTATTCAATTATAGTTATTAAAAATTAAAAATCATA
		TAATTTTTTAAATAACTTTTAGTGAACTAATCCTAAAGTTATCATT
		TTAATCTTGGAGGAATAAATTTAAACCCTAATCTAATTGGTTTATA
		TGTGTATTAACTAAATTACGAGATATTAGTTTTTGACACTTTTTTT
		CTCGTGGGTCGGCATGGCATCTCCACCTCCTCGCGGTCCGACCTGG
		GCATCCGAAGGAGGACGCACGTCCACTCGGATGGCTAAGGGAGAGC
		CTGCAGTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGG
		TTTTTTGGTGAAAGGAGGAACTATAGGCCGGCCCACCGCATATGTC
		TCAGTACAATCTGCTCTGATGCCGCATAGCCATCACATTGTACACG
		TGGACAAGTTGTCGCGGCCGCTTCTATAGTGTCACCTAAATACTAG
		TGACTCCAGCGTAACTGGACTGGCCACAGTTAGGCCGCAAATGTAA
		TCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTCGCGAGTGG
		ACCCGATAAGCTCATGGAGCGGCGTAACCGTCGCACAGGAAGGACA
		GAGAAAGCGCGGATCTGGGAAGTGACGGACAGAACGGTCAGGACCT
		GGATTGGGGAGGCGGTTGCCGCCGCTGCTGCTGACGGTGTGACGTT
		CTCTGTTCCGGTCACACCACATACGTTCCGCCATTCCTATGCGATG
		CACATGCTGTATGCCGGTATACCGCTGAAAGTTCTGCAAAGCCTGA
		TGGGACATAAGTCCATCAGTTCAACGGAAGTCTACACGAAGGTTTT
		TGCGCTGGATGTGGCTGCCCGGCACCGGGTGCAGTTTGCGATGCCG
		GAGTCTGATGCGGTTGCGATGCTGAAACAATTATCCTGAGAATAAA
		TGCCTTGGCCTTTATATGGAAATGTGGAACTGAGTGGATATGCTGT
		TTTTGTCTGTTAAACAGAGAAGCTGGCTGTTATCCACTGAGAAGCG
		AACGAAACAGTCGGGAAAATCTCCCATTATCGTAGAGATCCGCATT
		ATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTG
		TCATGATGCCTGCAAGCGGTAACGAAAACGATTTGAATATGCCTTC
		AGGAACAATAGAAATCTTCGTGCGGTGTTACGTTGAAGTGGAGCGG
		ATTATGTCAGCAATGGACAGAACAACCTAATGAACACAGAACCATG
		ATGTGGTCTGTCCTTTTACAGCCAGTAGTGCTCGCCGCAGTCGAGC
		GACAGGGCGAAGCCCTCGGCTGGTTGCCCTCGCCGCTGGGCTGGCG
		GCCGTCTATGGCCCTGCAAACGCGCCAGAAACGCCGTCGAAGCCGT
		GTGCGAGACACCGCGGCCGGCCGCCGGCGTTGTGGATACCTCGCGG
		AAAACTTGGCCCTCACTGACAGATGAGGGGCGGACGTTGACACTTG
		AGGGGCCGACTCACCCGGCGCGGCGTTGACAGATGAGGGGCAGGCT
		CGATTTCGGCCGGCGACGTGGAGCTGGCCAGCCTCGCAAATCGGCG
		AAAACGCCTGATTTTACGCGAGTTTCCCACAGATGATGTGGACAAG
		CCTGGGGATAAGTGCCCTGCGGTATTGACACTTGAGGGGCGCGACT
		ACTGACAGATGAGGGGCGCGATCCTTGACACTTGAGGGGCAGAGTG
		CTGACAGATGAGGGGCGCACCTATTGACATTTGAGGGGCTGTCCAC
		AGGCAGAAAATCCAGCATTTGCAAGGGTTTCCGCCCGTTTTTCGGC
		CACCGCTAACCTGTCTTTTAACCTGCTTTTAAACCAATATTTATAA
		ACCTTGTTTTTAACCAGGGCTGCGCCCTGTGCGCGTGACCGCGCAC
		GCCGAAGGGGGGTGCCCCCCCTTCTCGAACCCTCCCGGTCGAGTGA
		GCGAGGAAGCACCAGGGAACAGCACTTATATATTCTGCTTACACAC
		GATGCCTGAAAAAACTTCCCTTGGGGTTATCCACTTATCCACGGGG
		ATATTTTTATAATTATTTTTTTTATAGTTTTTAGATCTTCTTTTTT
		AGAGCGCCTTGTAGGCCTTTATCCATGCTGGTTCTAGAGAAGGTGT
		TGTGACAAATTGCCCTTTCAGTGTGACAAATCACCCTCAAATGACA
		GTCCTGTCTGTGACAAATTGCCCTTAACCCTGTGACAAATTGCCCT
		CAGAAGAAGCTGTTTTTTCACAAAGTTATCCCTGCTTATTGACTCT
		TTTTTATTTAGTGTGACAATCTAAAAACTTGTCACACTTCACATGG
		ATCTGTCATGGCGGAAACAGCGGTTATCAATCACAAGAAACGTAAA
		AATAGCCCGCGAATCGTCCAGTCAAACGACCTCACTGAGGCGGCAT
		ATAGTCTCTCCCGGGATCAAAAACGTATGCTGTATCTGTTCGTTGA
		CCAGATCAGAAAATCTGATGGCACCCTACAGGAACATGACGGTATC
		TGCGAGATCCATGTTGCTAAATATGCTGAAATATTCGGATTGACCT
		CTGCGGAAGCCAGTAAGGATATACGGCAGGCATTGAAGAGTTTCGC
		GGGGAAGGAAGTGGTTTTTTATCGCCCTGAAGAGGATGCCGGCGAT
		GAAAAAGGCTATGAATCTTTTCCTTGGTTTATCAAACGTGCGCACA
		GTCCATCCAGAGGGCTTTACAGTGTACATATCAACCCATATCTCAT
		TCCCTTCTTTATCGGGTTACAGAACCGGTTTACGCAGTTTCGGCTT
		AGTGAAACAAAAGAAATCACCAATCCGTATGCCATGCGTTTATACG
		AATCCCTGTGTCAGTATCGTAAGCCGGATGGCTCAGGCATCGTCTC
		TCTGAAAATCGACTGGATCATAGAGCGTTACCAGCTGCCTCAAAGT
		TACCAGCGTATGCCTGACTTCCGCCGCCGCTTCCTGCAGGTCTGTG
		TTAATGAGATCAACAGCAGAACTCCAATGCGCCTCTCATACATTGA
		GAAAAAGAAAGGCCGCCAGACGACTCATATCGTATTTTCCTTCCGC
		GATATCACTTCCATGACGACAGGATAGTCTGAGGGTTATCTGTCAC
		AGATTTGAGGGTGGTTCGTCACATTTGTTCTGACCTACTGAGGGTA
		ATTTGTCACAGTTTTGCTGTTTCCTTCAGCCTGCATGGATTTTCTC
		ATACTTTTTGAACTGTAATTTTTAAGGAAGCCAAATTTGAGGGCAG
		TTTGTCACAGTTGATTTCCTTCTCTTTCCCTTCGTCATGTGACCTG
		ATATCGGGGGTTAGTTCGTCATCATTGATGAGGGTTGATTATCACA
		GTTTATTACTCTGAATTGGCTATCCGCGTGTGTACCTCTACCTGGA
		GTTTTTCCCACGGTGGATATTTCTTCTTGCGCTGAGCGTAAGAGCT
		ATCTGACAGAACAGTTCTTCTTTGCTTCCTCGCCAGTTCGCTCGCT
		ATGCTCGGTTACACGGCTGCGGCGAGCGCTAGTGATAATAAGTGAC
		TGAGGTATGTGCTCTTCTTATCTCCTTTTGTAGTGTTGCTCTTATT
		TTAAACAACTTTGCGGTTTTTTGATGACTTTGCGATTTTGTTGTTG
		CTTTGCAGTAAATTGCAAGATTTAATAAAAAAACGCAAAGCAATGA
		TTAAAGGATGTTCAGAATGAAACTCATGGAAACACTTAACCAGTGC
		ATAAACGCTGGTCATGAAATGACGAAGGCTATCGCCATTGCACAGT
		TTAATGATGACAGCCCGGAAGCGAGGAAAATAACCCGGCGCTGGAG
		AATAGGTGAAGCAGCGGATTTAGTTGGGGTTTCTTCTCAGGCTATC
		AGAGATGCCGAGAAAGCAGGGCGACTACCGCACCCGGATATGGAAA
		TTCGAGGACGGGTTGAGCAACGTGTTGGTTATACAATTGAACAAAT
		TAATCATATGCGTGATGTGTTTGGTACGCGATTGCGACGTGCTGAA
		GACGTATTTCCACCGGTGATCGGGGTTGCTGCCCATAAAGGAGGCG
		TTTACAAAACCTCAGTTTCTGTTCATCTTGCTCAGGATCTGGCTCT
		GAAGGGGCTACGTGTTTTGCTCGTGGAAGGTAACGACCCCCAGGGA
		ACAGCCTCAATGTATCACGGATGGGTACCAGATCTTCATATTCATG
		CAGAAGACACTCTCCTGCCTTTCTATCTTGGGGAAAAGGACGATGT
		CACTTATGCAATAAAGCCCACTTGCTGGCCGGGGCTTGACATTATT
		CCTTCCTGTCTGGCTCTGCACCGTATTGAAACTGAGTTAATGGGCA
		AATTTGATGAAGGTAAACTGCCCACCGATCCACACCTGATGCTCCG
		ACTGGCCATTGAAACTGTTGCTCATGACTATGATGTCATAGTTATT
		GACAGCGCGCCTAACCTGGGTATCGGCACGATTAATGTCGTATGTG
		CTGCTGATGTGCTGATTGTTCCCACGCCTGCTGAGTIGTTTGACTA
		CACCTCCGCACTGCAGTTTTTCGATATGCTTCGTGATCTGCTCAAG
		AACGTTGATCTTAAAGGGTTCGAGCCTGATGTACGTATTTTGCTTA
		CCAAATACAGCAATAGTAATGGCTCTCAGTCCCCGTGGATGGAGGA
		GCAAATTCGGGATGCCTGGGGAAGCATGGTTCTAAAAAATGTTGTA
		CGTGAAACGGATGAAGTTGGTAAAGGTCAGATCCGGATGAGAACTG
		TTTTTGAACAGGCCATTGATCAACGCTCTTCAACTGGTGCCTGGAG
		AAATGCTCTTTCTATTTGGGAACCTGTCTGCAATGAAATTTTCGAT
		CGTCTGATTAAACCACGCTGGGAGATTAGATAATGAAGCGTGCGCC
		TGTTATTCCAAAACATACGCTCAATACTCAACCGGTTGAAGATACT
		TCGTTATCGACACCAGCTGCCCCGATGGTGGATTCGTTAATTGCGC
		GCGTAGGAGTAATGGCTCGCGGTAATGCCATTACTTTGCCTGTATG
		TGGTCGGGATGTGAAGTTTACTCTTGAAGTGCTCCGGGGTGATAGT
		GTTGAGAAGACCTCTCGGGTATGGTCAGGTAATGAACGTGACCAGG
		AGCTGCTTACTGAGGACGCACTGGATGATCTCATCCCTTCTTTTCT
		ACTGACTGGTCAACAGACACCGGCGTTCGGTCGAAGAGTATCTGGT
		GTCATAGAAATTGCCGATGGGAGTCGCCGTCGTAAAGCTGCTGCAC
		TTACCGAAAGTGATTATCGTGTTCTGGTTGGCGAGCTGGATGATGA
		GCAGATGGCTGCATTATCCAGATTGGGTAACGATTATCGCCCAACA
		AGTGCTTATGAACGTGGTCAGCGTTATGCAAGCCGATTGCAGAATG
		AATTTGCTGGAAATATTTCTGCGCTGGCTGATGCGGAAAATATTTC
		ACGTAAGATTATTACCCGCTGTATCAACACCGCCAAATTGCCTAAA
		TCAGTTGTTGCTCTTTTTTCTCACCCCGGTGAACTATCTGCCCGGT
		CAGGTGATGCACTTCAAAAAGCCTTTACAGATAAAGAGGAATTACT
		TAAGCAGCAGGCATCTAACCTTCATGAGCAGAAAAAAGCTGGGGTG
		ATATTTGAAGCTGAAGAAGTTATCACTCTTTTAACTTCTGTGCTTA
		AAACGTCATCTGCATCAAGAACTAGTTTAAGCTCACGACATCAGTT
		TGCTCCTGGAGCGACAGTATTGTATAAGGGCGATAAAATGGTGCTT
		AACCTGGACAGGTCTCGTGTTCCAACTGAGTGTATAGAGAAAATTG
		AGGCCATTCTTAAGGAACTTGAAAAGCCAGCACCCTGATGCGACCA
		CGTTTTAGTCTACGTTTATCTGTCTTTACTTAATGTCCTTTGTTAC
		AGGCCAGAAAGCATAACTGGCCTGAATATTCTCTCTGGGCCCACTG
		TTCCACTTGTATCGTCGGTCTGATAATCAGACTGGGACCACGGTCC
		CACTCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCAC
		TCGTATCGTCGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCG
		TATCGTCGGTCTGATAATCAGACTGGGACCACGGTCCCACTCGTAT
		CGTCGGTCTGATTATTAGTCTGGGACCATGGTCCCACTCGTATCGT
		CGGTCTGATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGG
		TCTGATTATTAGTCTGGAACCACGGTCCCACTCGTATCGTCGGTCT
		GATTATTAGTCTGGGACCACGGTCCCACTCGTATCGTCGGTCTGAT
		TATTAGTCTGGGACCACGATCCCACTCGTGTTGTCGGTCTGATTAT
		CGGTCTGGGACCACGGTCCCACTTGTATTGTCGATCAGACTATCAG
		CGTGAGACTACGATTCCATCAATGCCTGTCAAGGGCAAGTATTGAC
		ATGGTCGTCGTAACCTGTAGAACGGAGTAACCTCGGTGTGCGGTTG
		TATGCCTGCTGTGGATTGCTGCTGTGTCCTGCTTATCCACAACATT
		TTGCGCACGGTTATGTGGACAAAATACCTGGTTACCCAGGCCGTGC
		CGGCACGTTAACCGGGCTGCATCCGATGCAAGTGTGTCGCTGTCGA
		CGAGCTCGCGAGCTCGGACATGAGGTTGCCCCGTATTCAGTGTCGC
		TGATTTGTATTGTCTGAAGTTGCTTTTACGTTAAGTTGATGCAGAT
		CAATTAATACGATACCTGCGTCATAATTGATTATTTGACGTGGTTT
		GATGGCCTCCACGCACGTTGTGATATGTAGATGATAATCATTATCA
		CTTTACGGGTCCTTTCCGGTGATCCGACAGGTTACGGGGCGGCGAC
		CTCGCGGGTTTTCGCTATTTATGAAAATTTTCCGGTTTAAGGCGTT
		TCCGTTCTTCTTCGTCATAACTTAATGTTTTTATTTAAAATACCCT
		CTGAAAAGAAAGGAAACGACAGGTGCTGAAAGCGAGCTTTTTGGCC
		TCTGTCGTTTCCTTTCTCTGTTTTTGTCCGTGGAATGAACAATGGA
		AGTCCGAGCTCATCGCTAATAACTTCGTATAGCATACATTATACGA
		AGTTATATTCGATGCGGCCGATCTAGCAGAAAGTCAAAAGCCTCCG
		ACCGGAGGCTTTTGACTTCTGTCACCTAGGTTACGCCCCGCCCTGC
		CACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACAT
		GGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCAT
		CAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACG
		GGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGG
		TGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAAT
		AAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACA
		TCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATT
		CACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGT
		GTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTC
		ATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAA
		TGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGT
		CTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTA
		CATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCC
		ATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCAT
		TTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACG
		CCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTA
		CGTGCCGATCAGATTAAAACGAAAGGCCCAGTCTTTCGACTGAGCC
		TTTCGTTTTATTTGACCATGTTGGTATGATTTAAATTCAGTGCGGC
		CGCGACTTCAAGTCACGT
22	RSV A2	ATGGGCAGCAATTCATTGAGTATGATAAAAGTTAGATTACAAAATT
	NLucP	TGTTTGACAATGATGAAGTAGCATTGTTAAAAATAACATGCTATAC
	(without	TGATAAATTAATACATTTAACTAACGCTTTGGCTAAGGCAGTGATA
	vector)	CATACAATCAAATTGAATGGCATTGTGTTTGTGCATGTTATTACAA
	NS1 CDS: nt	GTAGTGATATTTGCCCTAATAATAATATTGTAGTAAAATCCAATTT
	1-417	CACAACAATGCCAGTACTACAAAATGGAGGTTATATATGGGAAATG
	NS2 CDS: nt	ATGGAATTAACACATTGCTCTCAACCTAATGGTCTACTAGATGACA
	530-901	ATTGTGAAATTAAATTCTCCAAAAAACTAAGTGATTCAACAATGAC
	N CDS: nt	CAATTATATGAATCAATTATCTGAATTACTTGGATTTGATCTTAAT
	1042-2214	CCATAAATTATAATTAATATCAACTAGCAAATCAATGTCACTAACA
	P CDS: nt	CCATTAGTTAATATAAAACTTAACAGAAGACAAAAATGGGGCAAAT
	2248-2970	AAATCAATTCAGCCAACCCAACCATGGACACAACCCACAATGATAA
	Intergenic P-M	TACACCACAAAGACTGATGATCACAGACATGAGACCGTTGTCACTT
	region: nt	GAGACCATAATAACATCACTAACCAGAGACATCATAACACACAAAT
	3145-3824	TTATATACTTGATAAATCATGAATGCATAGTGAGAAAACTTGATGA
	GS-Nluc-GE:	AAAACAGGCCACATTTACATTCCTGGTCAACTATGAAATGAAACTA
	nt 3149-3819	TTACACAAAGTAGGAAGCACTAAATATAAAAAATATACTGAATACA
	GS: nt	ACACAAAATATGGCACTTTCCCTATGCCAATATTCATCAATCATGA
	3149-3163	TGGGTTCTTAGAATGCATTGGCATTAAGCCTACAAAGCATACTCCC
	GE: nt	ATAATATACAAGTATGATCTCAATCCATAAATTTCAACACAATATT
	3803-3819	CACACAATCTAAAACAACAACTCTATGCATAACTATACTCCATAGT
	nanoLucP: nt	CCAGATGGAGCCTGAAAATTATAGTAATTTAAAATTAAGGAGAGAT
	3164-3802	ATAAGATAGAAGATGGGGCAAATACAAAGATGGCTCTTAGCAAAGT
	M CDS: nt	CAAGTTGAATGATACACTCAACAAAGATCAACTTCTGTCATCCAGC
	3834-4601	AAATACACCATCCAACGGAGCACAGGAGATAGTATTGATACTCCTA
	SH CDS: nt	ATTATGATGTGCAGAAACACATCAATAAGTTATGTGGCATGTTATT
	4876-5067	AATCACAGAAGATGCTAATCATAAATTCACTGGGTTAATAGGTATG
	G CDS: nt	TTATATGCGATGTCTAGGTTAGGAAGAGAAGACACCATAAAAATAC
	5261-6154	TCAGAGATGCGGGATATCATGTAAAAGCAAATGGAGTAGATGTAAC
	F CDS: nt	AACACATCGTCAAGACATTAATGGAAAAGAAATGAAATTTGAAGTG
	6234-7955	TTAACATTGGCAAGCTTAACAACTGAAATTCAAATCAACATTGAGA
	M2-1 CDS: nt	TAGAATCTAGAAAATCCTACAAAAAAATGCTAAAAGAAATGGGAGA
	8179-8760	GGTAGCTCCAGAATACAGGCATGACTCTCCTGATTGTGGGATGATA
	M2-2 CDS: nt	ATATTATGTATAGCAGCATTAGTAATAACTAAATTAGCAGCAGGGG
	8732-9001	ACAGATCTGGTCTTACAGCCGTGATTAGGAGAGCTAATAATGTCCT
	L CDS: nt	AAAAAATGAAATGAAACGTTACAAAGGCTTACTACCCAAGGACATA
	9071-15,565	GCCAACAGCTTCTATGAAGTGTTTGAAAAACATCCCCACTTTATAG
		ATGTTTTTGTTCATTTTGGTATAGCACAATCTTCTACCAGAGGTGG
		CAGTAGAGTTGAAGGGATTTTTGCAGGATTGTTTATGAATGCCTAT
		GGTGCAGGGCAAGTGATGTTACGGTGGGGAGTCTTAGCAAAATCAG
		TTAAAAATATTATGTTAGGACATGCTAGTGTGCAAGCAGAAATGGA
		ACAAGTTGTTGAGGTTTATGAATATGCCCAAAAATTGGGTGGTGAA
		GCAGGATTCTACCATATATTGAACAACCCAAAAGCATCATTATTAT
		CTTTGACTCAATTTCCTCACTTCTCCAGTGTAGTATTAGGCAATGC
		TGCTGGCCTAGGCATAATGGGAGAGTACAGAGGTACACCGAGGAAT
		CAAGATCTATATGATGCAGCAAAGGCATATGCTGAACAACTCAAAG
		AAAATGGTGTGATTAACTACAGTGTACTAGACTTGACAGCAGAAGA
		ACTAGAGGCTATCAAACATCAGCTTAATCCAAAAGATAATGATGTA
		GAGCTTTGAGTTAATAAAAAATGGGGCAAATAAATCATCATGGAAA
		AGTTTGCTCCTGAATTCCATGGAGAAGATGCAAACAACAGGGCTAC
		TAAATTCCTAGAATCAATAAAGGGCAAATTCACATCACCCAAAGAT
		CCCAAGAAAAAAGATAGTATCATATCTGTCAACTCAATAGATATAG
		AAGTAACCAAAGAAAGCCCTATAACATCAAATTCAACTATTATCAA
		CCCAACAAATGAGACAGATGATACTGCAGGGAACAAGCCCAATTAT
		CAAAGAAAACCTCTAGTAAGTTTCAAAGAAGACCCTACACCAAGTG
		ATAATCCCTTTTCTAAACTATACAAAGAAACCATAGAAACATTTGA
		TAACAATGAAGAAGAATCCAGCTATTCATACGAAGAAATAAATGAT
		CAGACAAACGATAATATAACAGCAAGATTAGATAGGATTGATGAAA
		AATTAAGTGAAATACTAGGAATGCTTCACACATTAGTAGTGGCAAG
		TGCAGGACCTACATCTGCTCGGGATGGTATAAGAGATGCCATGATT
		GGTTTAAGAGAAGAAATGATAGAAAAAATCAGAACTGAAGCATTAA
		TGACCAATGACAGATTAGAAGCTATGGCAAGACTCAGGAATGAGGA
		AAGTGAAAAGATGGCAAAAGACACATCAGATGAAGTGTCTCTCAAT
		CCAACATCAGAGAAATTGAACAACCTATTGGAAGGGAATGATAGTG
		ACAATGATCTATCACTTGAAGATTTCTGATTAGTTACCACTCTTCA
		CATCAACACACAATACCAACAGAAGACCAACAAACTAACCAACCCA
		ATCATCCAACCAAACATCCATCCGCCAATCAGCCAAACAGCCAACA
		AAACAACCAGCCAATCCAAAACTAACCACCCGGAAAAAATCTATAA
		TATAGTTACAAAAAAAGGAAGGGGCAAATAAATCAATGGTCTTCAC
		ACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAAC
		CTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGA
		ATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGG
		TGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAA
		GGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGG
		TGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTA
		TGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTAT
		TTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGA
		TCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGA
		GCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATC
		AACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGAATT
		CTCACGGCTTTCCGCCTGAGGTTGAAGAGCAAGCCGCCGGTACATT
		GCCTATGTCCTGCGCACAAGAAAGCGGTATGGACCGGCACCCAGCC
		GCTTGTGCTTCAGCTCGCATCAACGTCTAAATTATAGTAATTTAAA
		AAGGGTGGGGCAAATATGGAAACATACGTGAACAAGCTTCACGAAG
		GCTCCACATACACAGCTGCTGTTCAATACAATGTCTTAGAAAAAGA
		CGATGACCCTGCATCACTTACAATATGGGTGCCCATGTTCCAATCA
		TCTATGCCAGCAGATTTACTTATAAAAGAACTAGCTAATGTCAACA
		TACTAGTGAAACAAATATCCACACCCAAGGGACCTTCACTAAGAGT
		CATGATAAACTCAAGAAGTGCAGTGCTAGCACAAATGCCCAGCAAA
		TTTACCATATGCGCTAATGTGTCCTTGGATGAAAGAAGCAAACTAG
		CATATGATGTAACCACACCCTGTGAAATCAAGGCATGTAGTCTAAC
		ATGCCTAAAATCAAAAAATATGTTGACTACAGTTAAAGATCTCACT
		ATGAAGACACTCAACCCTACACATGATATTATTGCTTTATGTGAAT
		TTGAAAACATAGTAACATCAAAAAAAGTCATAATACCAACATACCT
		AAGATCCATCAGTGTCAGAAATAAAGATCTGAACACACTTGAAAAT
		ATAACAACCACTGAATTCAAAAATGCTATCACAAATGCAAAAATCA
		TCCCTTACTCAGGATTACTATTAGTCATCACAGTGACTGACAACAA
		AGGAGCATTCAAATACATAAAGCCACAAAGTCAATTCATAGTAGAT
		CTTGGAGCTTACCTAGAAAAAGAAAGTATATATTATGTTACCACAA
		ATTGGAAGCACACAGCTACACGATTTGCAATCAAACCCATGGAAGA
		TTAACCTTTTTCCTCTACATCAGTGTGTTAATTCATACAAACTTTC
		TACCTACATTCTTCACTTCACCATCACAATCACAAACACTCTGTGG
		TTCAACCAATCAAACAAAACTTATCTGAAGTCCCAGATCATCCCAA
		GTCATTGTTTATCAGATCTAGTACTCAAATAAGTTAATAAAAAATA
		TACACATGGGGCAAATAATCATTGGAGGAAATCCAACTAATCACAA
		TATCTGTTAACATAGACAAGTCCACACACCATACAGAATCAACCAA
		TGGAAAATACATCCATAACAATAGAATTCTCAAGCAAATTCTGGCC
		TTACTTTACACTAATACACATGATCACAACAATAATCTCTTTGCTA
		ATCATAATCTCCATCATGATTGCAATACTAAACAAACTTTGTGAAT
		ATAACGTATTCCATAACAAAACCTTTGAGTTACCAAGAGCTCGAGT
		CAACACATAGCATTCATCAATCCAACAGCCCAAAACAGTAACCTTG
		CATTTAAAAATGAACAACCCCTACCTCTTTACAACACCTCATTAAC
		ATCCCACCATGCAAACCACTATCCATACTATAAAGTAGTTAATTAA
		AAATAGTCATAACAATGAACTAGGATATCAAGACTAACAATAACAT
		TGGGGCAAATGCAAACATGTCCAAAAACAAGGACCAACGCACCGCT
		AAGACATTAGAAAGGACCTGGGACACTCTCAATCATTTATTATTCA
		TATCATCGTGCTTATATAAGTTAAATCTTAAATCTGTAGCACAAAT
		CACATTATCCATTCTGGCAATGATAATCTCAACTTCACTTATAATT
		GCAGCCATCATATTCATAGCCTCGGCAAACCACAAAGTCACACCAA
		CAACTGCAATCATACAAGATGCAACAAGCCAGATCAAGAACACAAC
		CCCAACATACCTCACCCAGAATCCTCAGCTTGGAATCAGTCCCTCT
		AATCCGTCTGAAATTACATCACAAATCACCACCATACTAGCTTCAA
		CAACACCAGGAGTCAAGTCAACCCTGCAATCCACAACAGTCAAGAC
		CAAAAACACAACAACAACTCAAACACAACCCAGCAAGCCCACCACA
		AAACAACGCCAAAACAAACCACCAAGCAAACCCAATAATGATTTTC
		ACTTTGAAGTGTTCAACTTTGTACCCTGCAGCATATGCAGCAACAA
		TCCAACCTGCTGGGCTATCTGCAAAAGAATACCAAACAAAAAACCA
		GGAAAGAAAACCACTACCAAGCCCACAAAAAAACCAACCCTCAAGA
		CAACCAAAAAAGATCCCAAACCTCAAACCACTAAATCAAAGGAAGT
		ACCCACCACCAAGCCCACAGAAGAGCCAACCATCAACACCACCAAA
		ACAAACATCATAACTACACTACTCACCTCCAACACCACAGGAAATC
		CAGAACTCACAAGTCAAATGGAAACCTTCCACTCAACTTCCTCCGA
		AGGCAATCCAAGCCCTTCTCAAGTCTCTACAACATCCGAGTACCCA
		TCACAACCTTCATCTCCACCCAACACACCACGCCAGTAGTTACTTA
		AAAACATATTATCACAAAAAGCCATGACCAACTTAAACAGAATCAA
		AATAAACTCTGGGGCAAATAACAATGGAGTTGCTAATCCTCAAAGC
		AAATGCAATTACCACAATCCTCACTGCAGTCACATTTTGTTTTGCT
		TCTGGTCAAAACATCACTGAAGAATTTTATCAATCAACATGCAGTG
		CAGTTAGCAAAGGCTATCTTAGTGCTCTGAGAACTGGTTGGTATAC
		CAGTGTTATAACTATAGAATTAAGTAATATCAAGGAAAATAAGTGT
		AATGGAACAGATGCTAAGGTAAAATTGATAAAACAAGAATTAGATA
		AATATAAAAATGCTGTAACAGAATTGCAGTTGCTCATGCAAAGCAC
		ACCACCAACAAACAATCGAGCCAGAAGAGAACTACCAAGGTTTATG
		AATTATACACTCAACAATGCCAAAAAAACCAATGTAACATTAAGCA
		AGAAAAGGAAAAGAAGATTTCTTGTTTTTTTGTTAGGTGTTGGATC
		TGCAATCGCCAGTGGCGTTGCTGTATCTAAGGTCCTGCACCTAGAA
		GGGGAAGTGAACAAGATCAAAAGTGCTCTACTATCCACAAACAAGG
		CTCTAGTCAGCTTATCAAATGGAGTTAGTGTCTTAACCAGCAAAGT
		GTTAGACCTCAAAAACTATATAGATAAACAATTGTTACCTATTGTG
		AACAAGCAAAGCTGCAGCATATCAAATATAGAAACTGTGATAGAGT
		TCCAACAAAAGAACAACAGACTACTAGAGATTACCAGGGAATTTAG
		TGTTAATGCAGGTGTAACTACACCTGTAAGCACTTACATGTTAACT
		AATAGTGAATTATTGTCATTAATCAATGATATGCCTATAACAAATG
		ATCAGAAAAAGTTAATGTCCAACAATGTTCAAATAGTTAGACAGCA
		AAGTTACTCTATCATGTCCATAATAAAAGAGGAAGTCTTAGCATAT
		GTAGTACAATTACCACTATATGGTGTTATAGATACACCCTGTTGGA
		AACTACACACATCCCCTCTATGTACAACCAACACAAAAGAAGGGTC
		CAACATCTGTTTAACAAGAACTGACAGAGGATGGTACTGTGACAAT
		GCAGGATCAGTATCTTTCTTCCCACAAGCTGAAACATGTAAAGTTC
		AATCAAATCGAGTATTTTGTGACACAATGAACAGTTTAACATTACC
		AAGTGAAATAAATCTCTGCAATGTTGACATATTCAACCCCAAATAT
		GATTGTAAAATTATGACTTCAAAAACAGATGTAAGCAGCTCCGTTA
		TCACATCTCTAGGAGCCATTGTGTCATGCTATGGCAAAACTAAATG
		TACAGCATCCAATAAAAATCGTGGAATCATAAAGACATTTTCTAAC
		GGGTGCGATTATGTATCAAATAAAGGGATGGACACTGTGTCTGTAG
		GTAACACATTATATTATGTAAATAAGCAAGAAGGTAAAAGTCTCTA
		TGTAAAAGGTGAACCAATAATAAATTTCTATGACCCATTAGTATTC
		CCCTCTGATGAATTTGATGCATCAATATCTCAAGTCAACGAGAAGA
		TTAACCAGAGCCTAGCATTTATTCGTAAATCCGATGAATTATTACA
		TAATGTAAATGCTGGTAAATCCACCACAAATATCATGATAACTACT
		ATAATTATAGTGATTATAGTAATATTGTTATCATTAATTGCTGTTG
		GACTGCTCTTATACTGTAAGGCCAGAAGCACACCAGTCACACTAAG
		CAAAGATCAACTGAGTGGTATAAATAATATTGCATTTAGTAACTAA
		ATAAAAATAGCACCTAATCATGTTCTTACAATGGTTTACTATCTGC
		TCATAGACAACCCATCTGTCATTGGATTTTCTTAAAATCTGAACTT
		CATCGAAACTCTCATCTATAAACCATCTCACTTACACTATTTAAGT
		AGATTCCTAGTTTATAGTTATATAAAACACAATTGAATGCCAGATT
		AACTTACCATCTGTAAAAATGAAAACTGGGGCAAATATGTCACGAA
		GGAATCCTTGCAAATTTGAAATTCGAGGTCATTGCTTAAATGGTAA
		GAGGTGTCATTTTAGTCATAATTATTTTGAATGGCCACCCCATGCA
		CTGCTTGTAAGACAAAACTTTATGTTAAACAGAATACTTAAGTCTA
		TGGATAAAAGTATAGATACCTTATCAGAAATAAGTGGAGCTGCAGA
		GTTGGACAGAACAGAAGAGTATGCTCTTGGTGTAGTTGGAGTGCTA
		GAGAGTTATATAGGATCAATAAACAATATAACTAAACAATCAGCAT
		GTGTTGCCATGAGCAAACTCCTCACTGAACTCAATAGTGATGATAT
		CAAAAAGCTGAGGGACAATGAAGAGCTAAATTCACCCAAGATAAGA
		GTGTACAATACTGTCATATCATATATTGAAAGCAACAGGAAAAACA
		ATAAACAAACTATCCATCTGTTAAAAAGATTGCCAGCAGACGTATT
		GAAGAAAACCATCAAAAACACATTGGATATCCATAAGAGCATAACC
		ATCAACAACCCAAAAGAATCAACTGTTAGTGATACAAATGACCATG
		CCAAAAATAATGATACTACCTGACAAATATCCTTGTAGTATAACTT
		CCATACTAATAACAAGTAGATGTAGAGTTACTATGTATAATCAAAA
		GAACACACTATATTTCAATCAAAACAACCCAAATAACCATATGTAC
		TCACCGAATCAAACATTCAATGAAATCCATTGGACCTCTCAAGAAT
		TGATTGACACAATTCAAATTTTTCTACAACATCTAGGTATTATTGA
		GGATATATATACAATATATATATTAGTGTCATAACACTCAATTCTA
		ACACTCACCACATCGTTACATTATTAATTCAAACAATTCAAGTTGT
		GGGACAAAATGGATCCCATTATTAATGGAAATTCTGCTAATGTTTA
		TCTAACCGATAGTTATTTAAAAGGTGTTATCTCTTTCTCAGAGTGT
		AATGCTTTAGGAAGTTACATATTCAATGGTCCTTATCTCAAAAATG
		ATTATACCAACTTAATTAGTAGACAAAATCCATTAATAGAACACAT
		GAATCTAAAGAAACTAAATATAACACAGTCCTTAATATCTAAGTAT
		CATAAAGGTGAAATAAAATTAGAAGAACCTACTTATTTTCAGTCAT
		TACTTATGACATACAAGAGTATGACCTCGTCAGAACAGATTGCTAC
		CACTAATTTACTTAAAAAGATAATAAGAAGAGCTATAGAAATAAGT
		GATGTCAAAGTCTATGCTATATTGAATAAACTAGGGCTTAAAGAAA
		AGGACAAGATTAAATCCAACAATGGACAAGATGAAGACAACTCAGT
		TATTACGACCATAATCAAAGATGATATACTTTCAGCTGTTAAAGAT
		AATCAATCTCATCTTAAAGCAGACAAAAATCACTCTACAAAACAAA
		AAGACACAATCAAAACAACACTCTTGAAGAAATTGATGTGTTCAAT
		GCAACATCCTCCATCATGGTTAATACATTGGTTTAACTTATACACA
		AAATTAAACAACATATTAACACAGTATCGATCAAATGAGGTAAAAA
		ACCATGGGTTTACATTGATAGATAATCAAACTCTTAGTGGATTTCA
		ATTTATTTTGAACCAATATGGTTGTATAGTTTATCATAAGGAACTC
		AAAAGAATTACTGTGACAACCTATAATCAATTCTTGACATGGAAAG
		ATATTAGCCTTAGTAGATTAAATGTTTGTTTAATTACATGGATTAG
		TAACTGCTTGAACACATTAAATAAAAGCTTAGGCTTAAGATGCGGA
		TTCAATAATGTTATCTTGACACAACTATTCCTTTATGGAGATTGTA
		TACTAAAGCTATTTCACAATGAGGGGTTCTACATAATAAAAGAGGT
		AGAGGGATTTATTATGTCTCTAATTTTAAATATAACAGAAGAAGAT
		CAATTCAGAAAACGATTTTATAATAGTATGCTCAACAACATCACAG
		ATGCTGCTAATAAAGCTCAGAAAAATCTGCTATCAAGAGTATGTCA
		TACATTATTAGATAAGACAGTGTCCGATAATATAATAAATGGCAGA
		TGGATAATTCTATTAAGTAAGTTCCTTAAATTAATTAAGCTTGCAG
		GTGACAATAACCTTAACAATCTGAGTGAACTATATTTTTTGTTCAG
		AATATTTGGACACCCAATGGTAGATGAAAGACAAGCCATGGATGCT
		GTTAAAATTAATTGCAATGAGACCAAATTTTACTTGTTAAGCAGTC
		TGAGTATGTTAAGAGGTGCCTTTATATATAGAATTATAAAAGGGTT
		TGTAAATAATTACAACAGATGGCCTACTTTAAGAAATGCTATTGTT
		TTACCCTTAAGATGGTTAACTTACTATAAACTAAACACTTATCCTT
		CTTTGTTGGAACTTACAGAAAGAGATTTGATTGTGTTATCAGGACT
		ACGTTTCTATCGTGAGTTTCGGTTGCCTAAAAAAGTGGATCTTGAA
		ATGATTATAAATGATAAAGCTATATCACCTCCTAAAAATTTGATAT
		GGACTAGTTTCCCTAGAAATTACATGCCATCACACATACAAAACTA
		TATAGAACATGAAAAATTAAAATTTTCCGAGAGTGATAAATCAAGA
		AGAGTATTAGAGTATTATTTAAGAGATAACAAATTCAATGAATGTG
		ATTTATACAACTGTGTAGTTAATCAAAGTTATCTCAACAACCCTAA
		TCATGTGGTATCATTGACAGGCAAAGAAAGAGAACTCAGTGTAGGT
		AGAATGTTTGCAATGCAACCGGGAATGTTCAGACAGGTTCAAATAT
		TGGCAGAGAAAATGATAGCTGAAAACATTTTACAATTCTTTCCTGA
		AAGTCTTACAAGATATGGTGATCTAGAACTACAAAAAATATTAGAA
		TTGAAAGCAGGAATAAGTAACAAATCAAATCGCTACAATGATAATT
		ACAACAATTACATTAGTAAGTGCTCTATCATCACAGATCTCAGCAA
		ATTCAATCAAGCATTTCGATATGAAACGTCATGTATTTGTAGTGAT
		GTGCTGGATGAACTGCATGGTGTACAATCTCTATTTTCCTGGTTAC
		ATTTAACTATTCCTCATGTCACAATAATATGCACATATAGGCATGC
		ACCCCCCTATATAGGAGATCATATTGTAGATCTTAACAATGTAGAT
		GAACAAAGTGGATTATATAGATATCACATGGGTGGCATCGAAGGGT
		GGTGTCAAAAACTGTGGACCATAGAAGCTATATCACTATTGGATCT
		AATATCTCTCAAAGGGAAATTCTCAATTACTGCTTTAATTAATGGT
		GACAATCAATCAATAGATATAAGCAAACCAATCAGACTCATGGAAG
		GTCAAACTCATGCTCAAGCAGATTATTTGCTAGCATTAAATAGCCT
		TAAATTACTGTATAAAGAGTATGCAGGCATAGGCCACAAATTAAAA
		GGAACTGAGACTTATATATCACGAGATATGCAATTTATGAGTAAAA
		CAATTCAACATAACGGTGTATATTACCCAGCTAGTATAAAGAAAGT
		CCTAAGAGTGGGACCGTGGATAAACACTATACTTGATGATTTCAAA
		GTGAGTCTAGAATCTATAGGTAGTTTGACACAAGAATTAGAATATA
		GAGGTGAAAGTCTATTATGCAGTTTAATATTTAGAAATGTATGGTT
		ATATAATCAGATTGCTCTACAATTAAAAAATCATGCATTATGTAAC
		AATAAACTATATTTGGACATATTAAAGGTTCTGAAACACTTAAAAA
		CCTTTTTTAATCTTGATAATATTGATACAGCATTAACATTGTATAT
		GAATTTACCCATGTTATTTGGTGGTGGTGATCCCAACTTGTTATAT
		CGAAGTTTCTATAGAAGAACTCCTGACTTCCTCACAGAGGCTATAG
		TTCACTCTGTGTTCATACTTAGTTATTATACAAACCATGACTTAAA
		AGATAAACTTCAAGATCTGTCAGATGATAGATTGAATAAGTTCTTA
		ACATGCATAATCACGTTTGACAAAAACCCTAATGCTGAATTCGTAA
		CATTGATGAGAGATCCTCAAGCTTTAGGGTCTGAGAGACAAGCTAA
		AATTACTAGCGAAATCAATAGACTGGCAGTTACAGAGGTTTTGAGT
		ACAGCTCCAAACAAAATATTCTCCAAAAGTGCACAACATTATACTA
		CTACAGAGATAGATCTAAATGATATTATGCAAAATATAGAACCTAC
		ATATCCTCATGGGCTAAGAGTTGTTTATGAAAGTTTACCCTTTTAT
		AAAGCAGAGAAAATAGTAAATCTTATATCAGGTACAAAATCTATAA
		CTAACATACTGGAAAAAACTTCTGCCATAGACTTAACAGATATTGA
		TAGAGCCACTGAGATGATGAGGAAAAACATAACTTTGCTTATAAGG
		ATACTTCCATTGGATTGTAACAGAGATAAAAGAGAGATATTGAGTA
		TGGAAAACCTAAGTATTACTGAATTAAGCAAATATGTTAGGGAAAG
		ATCTTGGTCTTTATCCAATATAGTTGGTGTTACATCACCCAGTATC
		ATGTATACAATGGACATCAAATATACTACAAGCACTATATCTAGTG
		GCATAATTATAGAGAAATATAATGTTAACAGTTTAACACGTGGTGA
		GAGAGGACCCACTAAACCATGGGTTGGTTCATCTACACAAGAGAAA
		AAAACAATGCCAGTTTATAATAGACAAGTCTTAACCAAAAAACAGA
		GAGATCAAATAGATCTATTAGCAAAATTGGATTGGGTGTATGCATC
		TATAGATAACAAGGATGAATTCATGGAAGAACTCAGCATAGGAACC
		CTTGGGTTAACATATGAAAAGGCCAAGAAATTATTTCCACAATATT
		TAAGTGTCAATTATTTGCATCGCCTTACAGTCAGTAGTAGACCATG
		TGAATTCCCTGCATCAATACCAGCTTATAGAACAACAAATTATCAC
		TTTGACACTAGCCCTATTAATCGCATATTAACAGAAAAGTATGGTG
		ATGAAGATATTGACATAGTATTCCAAAACTGTATAAGCTTTGGCCT
		TAGTTTAATGTCAGTAGTAGAACAATTTACTAATGTATGTCCTAAC
		AGAATTATTCTCATACCTAAGCTTAATGAGATACATTTGATGAAAC
		CTCCCATATTCACAGGTGATGTTGATATTCACAAGTTAAAACAAGT
		GATACAAAAACAGCATATGTTTTTACCAGACAAAATAAGTTTGACT
		CAATATGTGGAATTATTCTTAAGTAATAAAACACTCAAATCTGGAT
		CTCATGTTAATTCTAATTTAATATTGGCACATAAAATATCTGACTA
		TTTTCATAATACTTACATTTTAAGTACTAATTTAGCTGGACATTGG
		ATTCTGATTATACAACTTATGAAAGATTCTAAAGGTATTTTTGAAA
		AAGATTGGGGAGAGGGATATATAACTGATCATATGTTTATTAATTT
		GAAAGTTTTCTTCAATGCTTATAAGACCTATCTCTTGTGTTTTCAT
		AAAGGTTATGGCAAAGCAAAGCTGGAGTGTGATATGAACACTTCAG
		ATCTTCTATGTGTATTGGAATTAATAGACAGTAGTTATTGGAAGTC
		TATGTCTAAGGTATTTTTAGAACAAAAAGTTATCAAATACATTCTT
		AGCCAAGATGCAAGTTTACATAGAGTAAAAGGATGTCATAGCTTCA
		AATTATGGTTTCTTAAACGTCTTAATGTAGCAGAATTCACAGTTTG
		CCCTTGGGTTGTTAACATAGATTATCATCCAACACATATGAAAGCA
		ATATTAACTTATATAGATCTTGTTAGAATGGGATTGATAAATATAG
		ATAGAATACACATTAAAAATAAACACAAATTCAATGATGAATTTTA
		TACTTCTAATCTCTTCTACATTAATTATAACTTCTCAGATAATACT
		CATCTATTAACTAAACATATAAGGATTGCTAATTCTGAATTAGAAA
		ATAATTACAACAAATTATATCATCCTACACCAGAAACCCTAGAGAA
		TATACTAGCCAATCCGATTAAAAGTAATGACAAAAAGACACTGAAT
		GACTATTGTATAGGTAAAAATGTTGACTCAATAATGTTACCATTGT
		TATCTAATAAGAAGCTTATTAAATCGTCTGCAATGATTAGAACCAA
		TTACAGCAAACAAGATTTGTATAATTTATTCCCTATGGTTGTGATT
		GATAGAATTATAGATCATTCAGGCAATACAGCCAAATCCAACCAAC
		TTTACACTACTACTTCCCACCAAATATCTTTAGTCCACAATAGCAC
		ATCACTTTACTGCATGCTTCCTTGGCATCATATTAATAGATTCAAT
		TTTGTATTTAGTTCTACAGGTTGTAAAATTAGTATAGAGTATATTT
		TAAAAGATCTTAAAATTAAAGATCCCAATTGTATAGCATTCATAGG
		TGAAGGAGCAGGGAATTTATTATTGCGTACAGTAGTGGAACTTCAT
		CCTGACATAAGATATATTTACAGAAGTCTGAAAGATTGCAATGATC
		ATAGTTTACCTATTGAGTTTTTAAGGCTGTACAATGGACATATCAA
		CATTGATTATGGTGAAAATTTGACCATTCCTGCTACAGATGCAACC
		AACAACATTCATTGGTCTTATTTACATATAAAGTTTGCTGAACCTA
		TCAGTCTTTTTGTCTGTGATGCCGAATTGTCTGTAACAGTCAACTG
		GAGTAAAATTATAATAGAATGGAGCAAGCATGTAAGAAAGTGCAAG
		TACTGTTCCTCAGTTAATAAATGTATGTTAATAGTAAAATATCATG
		CTCAAGATGATATTGATTTCAAATTAGACAATATAACTATATTAAA
		AACTTATGTATGCTTAGGCAGTAAGTTAAAGGGATCGGAGGTTTAC
		TTAGTCCTTACAATAGGTCCTGCGAATATATTCCCAGTATTTAATG
		TAGTACAAAATGCTAAATTGATACTATCAAGAACCAAAAATTTCAT
		CATGCCTAAGAAAGCTGATAAAGAGTCTATTGATGCAAATATTAAA
		AGTTTGATACCCTTTCTTTGTTACCCTATAACAAAAAAAGGAATTA
		ATACTGCATTGTCAAAACTAAAGAGTGTTGTTAGTGGAGATATACT
		ATCATATTCTATAGCTGGACGTAATGAAGTTTTCAGCAATAAACTT
		ATAAATCATAAGCATATGAACATCTTAAAATGGTTCAATCATGTTT
		TAAATTTCAGATCAACAGAACTAAACTATAACCATTTATATATGGT
		AGAATCTACATATCCTTACCTAAGTGAATTGTTAAACAGCTTGACA
		ACCAATGAACTTAAAAAACTGATTAAAATCACAGGTAGTCTGTTAT
		ACAACTTTCATAATGAA

All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. § 1.57(b)(1), to relate to each and every individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. § 1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. To the extent that the references provide a definition for a claimed term that conflicts with the definitions provided in the instant specification, the definitions provided in the instant specification shall be used to interpret the claimed invention.

Claims

1. An expression vector for producing an infectious recombinant respiratory syncytial virus (RSV) comprising:

a) a nucleic acid sequence encoding a respiratory syncytial virus; and

b) a reporter gene flanked by an RSV gene start sequence and an RSV gene end sequence, the reporter gene and flanking RSV gene start and RSV gene end sequences located between the P and M genes of the respiratory syncytial virus;

wherein the RSV gene start sequence is SEQ ID NO: 16 and the RSV gene end sequence is SEQ ID NO: 17; and

wherein the reporter gene encodes a luminescent enzyme that catalyzes a luminescent substrate; or the reporter gene encodes a fluorescent protein.

2.-4. (canceled)

5. The expression vector of claim 1, wherein the sequence of the reporter gene is SEQ ID NO: 18.

6. The expression vector of claim 1, wherein the reporter gene flanked by the RSV gene start and gene end sequences is SEQ ID NO: 15.

7. The expression vector of claim 1, wherein the respiratory syncytial virus is strain A2 or comprises SEQ ID NO: 14.

8. A respiratory syncytial virus comprising:

a) a nucleic acid sequence encoding a respiratory syncytial virus; and

b) a reporter gene flanked by an RSV gene start sequence and an RSV gene end sequence, the reporter gene and flanking RSV gene start and RSV gene end sequences located between the P and M genes of the respiratory syncytial virus;

wherein the RSV gene start sequence is SEQ ID NO: 16 and the RSV gene end sequence is SEQ ID NO: 17; and

wherein the reporter gene encodes a luminescent enzyme that catalyzes a luminescent substrate; or the reporter gene encodes a fluorescent protein.

9.-11. (canceled)

12. The respiratory syncytial virus of claim 8, wherein the sequence of the reporter gene is SEQ ID NO: 18.

13. The respiratory syncytial virus of claim 8, wherein the reporter gene flanked by the RSV gene start and gene end sequences is SEQ ID NO: 15.

14. The respiratory syncytial virus of claim 8, wherein the respiratory syncytial virus is strain A2 or comprises SEQ ID NO: 14.

15. A method for measuring the activity of an anti-respiratory syncytial virus (RSV) antibody or antigen binding fragment thereof, the method comprising the steps of:

a) combining (i) the anti-RSV antibody or antigen binding fragment thereof, (ii) an RSV virus comprising a reporter gene, and (iii) one or more cells infectable by the RSV virus; and

b) detecting expression of the reporter gene;

wherein the RSV virus comprises i) a nucleic acid sequence encoding a respiratory syncytial virus; and ii) the reporter gene flanked by an RSV gene start sequence and an RSV gene end sequence, the reporter gene and flanking RSV gene start and RSV gene end sequences located between the P and M genes of the respiratory syncytial virus, wherein the reporter gene encodes a luminescent enzyme that catalyzes a luminescent substrate, and detecting expression of the reporter gene comprises detecting luminescence of the luminescent substrate, or wherein the reporter gene encodes a fluorescent protein, and detecting expression of the reporter gene comprises detecting fluorescent light emission from the fluorescent protein.

16. The method of claim 15, wherein the anti-RSV antibody or antigen binding fragment thereof of (i) is combined with the RSV virus (ii) before adding the combined mixture of (i) and (ii) to the one or more cells infectable by the RSV virus of (iii).

17. (canceled)

18. The method of claim 15, wherein the luminescent enzyme is a luciferase.

19. The method of claim 18, further comprising a step of adding a luciferase substrate.

20. The method of claim 15, wherein the one or more cells infectable by the RSV virus are A549 cells.

21. (canceled)

22. The method of claim 18, wherein the nucleic acid sequence encoding the luciferase encodes a nanoluciferase.

23. The method of claim 15, wherein the anti-RSV antibody or antigen binding fragment thereof comprises:

(a) three heavy chain complementarity determining regions (HC-CDRs), wherein HC-CDR1 is SEQ ID NO: 1, HC-CDR2 is SEQ ID NO: 2, and HC-CDR3 is SEQ ID NO: 3; and

(b) three light chain complementarity determining regions (LC-CDRs), wherein LC-CDR1 is SEQ ID NO: 4, LC-CDR2 is SEQ ID NO: 5, and LC-CDR3 is SEQ ID NO: 6.

24. The method of claim 15, wherein the anti-RSV antibody or antigen binding fragment thereof comprises a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8.

25. The method of claim 15, wherein the anti-RSV antibody or antigen binding fragment thereof comprises a heavy chain and a light chain, and wherein the heavy chain comprises SEQ ID NO: 9 and the light chain comprises SEQ ID NO: 10.

26. The method of claim 15, wherein the anti-RSV antibody or antigen binding fragment thereof is an antibody comprising two heavy chains of SEQ ID NO: 9 and two light chains of SEQ ID NO: 10.

27.-30. (canceled)

31. The method of claim 27, wherein the sequence of the reporter gene is SEQ ID NO: 18.

32. The method of claim 27, wherein the reporter gene flanked by an RSV gene start sequence and an RSV gene end sequence is SEQ ID NO: 15.

33. The method of claim 27, wherein the respiratory syncytial virus is strain A2 or comprises SEQ ID NO: 14.